Evolution of bond-forming enzymes

ABSTRACT

Strategies, systems, methods, reagents, and kits for the directed evolution of bond-forming enzymes are provided herein. Evolution products, for example, evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are also provided herein, as are methods for using such evolved bond-forming enzymes. Kits comprising materials, reagents, and cells for carrying out the directed evolution methods described herein are also provided.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.provisional application, U.S. Ser. No. 61/662,606, filed Jun. 21, 2012,the entire contents of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under grant R01GM065400 awarded by the National Institutes of Health and under grantHR0011-08-0085 awarded by United States Department of Defense and theDefense Advanced Research Projects Agency (DARPA). The U.S. Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The ability to routinely generate efficient enzymes that catalyzebond-forming reactions chosen by researchers, rather than nature, is along-standing goal of the molecular life sciences. Such catalysts can beused to form bonds between molecules, e.g., proteins, nucleic acids,carbohydrates, or small molecules, under physiological conditions, thusallowing in vivo and in vitro modification of molecules in or on livingcells and other biological structures while maintaining their structuralintegrity. The spectrum of bond-forming reactions catalyzed by naturallyoccurring enzymes, e.g., naturally occurring sortases, ligases,polymerases, and kinases, is limited and typically restricted tospecific substrates. For example, sortases catalyze a transpeptidationreaction that results in the conjugation of a peptide comprising aC-terminal sortase recognition motif with a peptide comprising anN-terminal sortase recognition motif. Naturally occurring sortases aretypically selective for specific C-terminal and N-terminal recognitionmotifs, e.g., LPXTG (SEQ ID NO: 51) (where X represents any amino acid)and GGG, respectively. The spectrum of peptides and proteins that can beconjugated via sortases is, therefore, limited. While target proteinsnot comprising a sortase recognition sequence may be engineered to addsuch a sequence, such engineering is often cumbersome or impractical,e.g., in situations where the addition of an exogenous sortaserecognition motif would disturb the structure and/or the function of thenative protein. Another obstacle to a broader application ofbond-forming enzymes to biological systems is that naturally occurringbond-forming enzymes typically exhibit low reaction efficiencies. Thegeneration of bond-forming enzymes that efficiently catalyzebond-forming reactions and/or utilize a desired target substrate, e.g.,a desired sortase recognition sequence, would allow for a broaderapplication of bond-forming reactions to conjugate biomolecules.

SUMMARY OF THE INVENTION

Provided herein are strategies, systems, methods, and reagents forevolving enzymes that catalyze any bond-forming reaction. The technologyprovided herein integrates yeast display, enzyme-mediatedbioconjugation, and fluorescence-activated cell sorting to isolate cellsexpressing proteins that catalyze the coupling of two target substrates.The strategies provided herein can be used to evolve bond-formingenzymes with improved catalytic activity and/or altered substratepreference. For example, as described herein, several variants of S.aureus sortase A were evolved that exhibited up to a 140-fold increasein transpeptidation activity compared to the starting wild type enzyme.One advantage of the evolution strategies provided herein is that theydo not rely on any particular screenable or selectable property of thesubstrates or reaction products. Accordingly, the evolution strategiesprovided herein are broadly applicable to evolve bond-forming enzymesutilizing any substrate and catalyzing any bond-forming reaction.

Some embodiments of this invention provide evolved sortases. In someembodiments, a sortase is provided that comprises an amino acid sequencethat is at least 90% homologous to the amino acid sequence of S. aureusSortase A as provided as SEQ ID NO: 1, or a fragment thereof. In someembodiments, the amino acid sequence of the sortase comprises one ormore mutations selected from the group consisting of P94S, P94R, E106G,F122Y, F154R, D160N, D165A, G174S, K190E, and K196T. In someembodiments, the sortase comprises an amino acid sequence that is atleast 95%, at least 98%, or at least 99% homologous to SEQ ID NO: 1, ora fragment thereof. In some embodiments, the amino acid sequence of thesortase comprises at least one mutation, at least two mutations, atleast three mutations, or at least four mutations as compared to theamino acid sequence of S. aureus Sortase A provided as SEQ ID NO: 1, ora fragment thereof. In some embodiments, wherein the sortase comprises aP94S or P94R mutation, a D160N mutation, a D165A mutation, a K190Emutation, and a K196T mutation. In some embodiments, the sortasecomprises a P94S or P94R mutation, a D160N mutation, and a K196Tmutation. In some embodiments, the sortase comprises a P94S or P94Rmutation, a D160N mutation, and a D165A mutation. In some embodiments,the sortase comprises a P94S or P94R mutation, a D160N mutation, a D165Amutation, and a K196T mutation. In some embodiments, the sortaseexhibits a k_(cat) that is at least 1.5-fold, at least 2-fold, or atleast 3-fold greater than the k_(cat) of the corresponding wild type S.aureus Sortase A. In some embodiments, the sortase exhibits a K_(M) fora substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32)that is at least 2-fold, at least 5-fold, or at least 10-fold less thanthe K_(M) of the corresponding wild type sortase A. In some embodiments,the sortase exhibits a K_(M) for a substrate comprising the amino acidsequence GGG that is not more than 2-fold, not more than 5-fold, notmore than 10-fold, or not more than 20-fold greater than the K_(M) ofthe corresponding wild type sortase A amino acid sequence. In someembodiments, the sortase exhibits a ratio of K_(cat)/k₄ for a substratecomprising the amino acid sequence LPETG (SEQ ID NO: 32) that is least2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, or at least 120-fold greater than theK_(cat)/K_(M) ratio of the corresponding wild type sortase A.

Certain embodiments of this invention provide evolved sortasescatalyzing transpeptidation reactions that utilize a substratecomprising a C-terminal sortase recognition sequence other than LPETG(SEQ ID NO: 32). In some embodiments, the sortase comprising an aminoacid sequence that is at least 90%, at least 95%, at least 98%, at least99%, or at least 99.5% homologous to the amino acid sequence of S.aureus Sortase A as provided as SEQ ID NO: 1 or a fragment thereof. Insome embodiments, the amino acid sequence comprises one or moremutations selected from the group consisting of P86L, N98S, A104T,A118T, F122S, D124G, N127S, K134R, K173E, K177E and 1182V. In someembodiments, the substrate of the evolved sortase comprises the aminoacid sequence LPXSX, wherein each X represents independently any aminoacid residue, for example, the amino acid sequence LPESG (SEQ ID NO:38).

In certain embodiments, this invention provides methods fortranspeptidation using an evolved sortase as described herein. In someembodiments, the method comprises contacting a sortase as describedherein with a substrate comprising a C-terminal LPETG (SEQ ID NO: 32)sequence and with a substrate comprising an N-terminal GGG sequenceunder conditions suitable for sortase-mediated transpeptidation. In someembodiments, the LPETG (SEQ ID NO: 32) substrate and/or the GGGsubstrate are on the surface of a cell. In some embodiments, the cellexpresses a surface marker protein that is C-terminally fused to anLPETG (SEQ ID NO: 32) sequence. In some embodiments, the cell expressesa surface marker protein that is N-terminally fused to a GGG sequence.In some embodiments, the LPETG (SEQ ID NO: 32) substrate and/or the GGGsubstrate are polypeptides or proteins, and the method results in thegeneration of a protein fusion. In some embodiments, the LPETG (SEQ IDNO: 32) substrate or the GGG substrate comprises a non-proteinstructure. In some embodiments, the LPETG (SEQ ID NO: 32) substrate orthe GGG substrate comprises a detectable label, a small molecule, anucleic acid, or a polysaccharide.

This invention also provides methods for directed evolution ofbond-forming enzymes. In some embodiments, the method comprises (a)providing a cell population in which (i) a first cell surface protein ora cell in the cell population is conjugated to a candidate bond-formingenzyme, wherein different cells within the population of cells comprisedifferent candidate bond-forming enzymes conjugated to the cell surfaceprotein and (ii) a second cell surface protein is conjugated to asubstrate A; (b) contacting the cell population with a substrate Bconjugated to a detectable label under conditions suitable for thebond-forming enzyme to form a bond between substrate A and substrate B;and (c) identifying and/or isolating a cell that is conjugated tosubstrate B. In some embodiments, the method comprises (a) providing ayeast cell population in which (i) a library of candidate bond-formingenzymes is expressed as a fusion to an Aga2p cell surface mating factor,wherein different cells within the cell population express differentcandidate bond-forming enzymes; (ii) an Aga1p cell surface mating factoris covalently bound to the Aga2p cell surface mating factor, wherein theAga2p cell surface mating factor is conjugated to a substrate A; (b)contacting the cell population with a substrate B conjugated to adetectable label under conditions suitable for the bond-forming enzymeto form a bond between substrate A and substrate B; and (c) identifyingand/or isolating a cell that is conjugated to substrate B. In someembodiments, the method further comprises (d) identifying and/orisolating the bond-forming enzyme(s) expressed in the cells isolated instep (c). In some embodiments, the method further comprises (e)subjecting the bond-forming enzyme(s) expressed in the cells isolated instep (c) to a diversification procedure, thus creating a diversifiedlibrary of candidate bond-forming enzymes, expressing the diversifiedcandidate bond-forming enzyme library as a fusion to an Aga2p cellsurface mating factor in a population of yeast cells, and repeatingsteps (a)-(c). In some embodiments, the diversification procedurecomprises random mutagenesis and/or recombination. In some embodiments,substrate A is conjugated to the cell surface protein or the Agap2 cellsurface mating factor via a reactive handle. In some embodiments, thecandidate enzyme is fused to the cell surface protein or the Agap1 cellsurface mating factor via a cleavable linker. In some embodiments, thecleavable linker comprises a protease cleavage site. In someembodiments, the method comprises multiple rounds of performing steps(a)-(d) and a final round of performing steps (a)-(c). In someembodiments, the method comprises decreasing the concentration ofsubstrate B in subsequent rounds of performing steps (a)-(d) or steps(a)-(c). In some embodiments, the method comprises using a modifiedsubstrate A or a modified substrate B in subsequent rounds of performingsteps (a)-(d) or steps (a)-(c). In some embodiments, the method furthercomprises comparing the bond-forming properties of at least one enzymeidentified or isolated in step (c) with the corresponding wild typeenzyme, wherein if the enzyme isolated in step (c) exhibits an improvedbond-forming characteristic, it is identified as an enhanced, evolvedbond-forming enzyme. In some embodiments, the bond-forming enzymes aretranspeptidases. In some embodiments, the transpeptidases are sortases.In some embodiments, the bond-forming enzymes are ligases (e.g., biotinligases, ubiquitin ligases, peptide ligases, subtiligases), polymerases,kinases, aldolases, diels alderases, transferases (e.g., biotinyltransferases, farnesyl transferases, or phosphopantathienyltransferases).

Other advantages, features, and uses of the invention will be apparentfrom the Detailed Description of Certain Embodiments, the Drawings, theExamples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A general strategy for the evolution of bond-forming catalystsusing yeast display.

FIG. 2. Validation of the enzyme evolution strategy. (A) FACS histogramof the reaction between cell surface-conjugated LPETGG (SEQ ID NO: 33)and free GGGYK-biotin (SEQ ID NO: 37) catalyzed by yeast-displayed wildtype S. aureus sortase A (wild type srtA). Cells were stained withstreptavidin-PE and an AlexaFluor488-anti-HA antibody. Negative controlreactions with either the inactive C184A srtA mutant or without LPETGG(SEQ ID NO: 33) are shown. (B) Dot plots comparing PE fluorescence(extent of reaction) vs. AlexaFluor488 fluorescence (display level) fortwo model screens. Mixtures of cells displaying either wild type srtA orthe inactive C184A srtA (1:1;000 and 1:100 wild type:C184A) wereprocessed as in A, then analyzed by FACS. Cells within the specifiedgate (black polygon) were collected. (C) Model screening results. Genecompositions before and after sorting were compared following HindIIIdigestion, revealing strong enrichment for active sortase.

FIG. 3. Activity assays of mutant sortases. (A) Yeast pools recoveredfrom the sorts were treated with TEV protease, and the cleaved enzymeswere assayed for their ability to catalyze coupling between 5 μMCoA-LPETGG (SEQ ID NO: 33) and 25 μM GGGYK-biotin (SEQ ID NO: 37). (B)Yeast cells expressing select individual clones were treated asdescribed in the Examples section. Error bars represent the standarddeviation of three independent experiments.

FIG. 4. Mutations in evolved sortases. (A) Highly enriched mutations arehighlighted in black; other mutations are shown in blue. (B) Mappingevolved mutations on the solution structure of wild type S. aureussortase A covalently bound to its Cbz-LPAT (SEQ ID NO: 39) substrate.The calcium ion is shown in blue, the LPAT (SEQ ID NO: 39) peptide iscolored cyan with red labels, and the side chains of amino acids thatare mutated are in orange. The N-terminal Cbz group is shown in stickform in cyan.

FIG. 5. Cell-surface labeling with wild type and mutant sortases. LiveHeLa cells expressing human CD154 conjugated at its extracellular Cterminus to LPETG (SEQ ID NO: 32) were incubated with 1 mM GGGYK-biotin(SEQ ID NO: 37) and no sortase A (srtA), 100 μM wild type srtA, or 100μM P94S/D160N/K196T srtA. The cells were stained withAlexa-Fluor-conjugated streptavidin. (A) Flow cytometry analysiscomparing cell labeling with wild type sortase (blue) and the mutantsortase (red). Negative control reactions omitting sortase (black) orLPETG (SEQ ID NO:32) (green) are shown. (B) Live cell confocalfluorescence microscopy images of cells. The yellow fluorescent protein(YFP, transfection marker) and Alexa (cell labeling) channels are shown.

FIG. 6. Sfp-catalyzed transfer of phosphopantetheinyl derivatives (blue)onto a specific serine residue (underlined) within the S6 peptidesequence (SEQ ID NO: 53).

FIG. 7. Engineering a Saccharomyces cerevisiae strain thatsimultaneously displays the S6 peptide sequence and the sortase libraryon its cell surface. The S6-Aga1p construct is cloned under the controlof the constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD)promoter and integrated into the genome of S. cerevisiae BJ5465 to yieldstrain ICY200. Through several cloning steps, a TEV recognition site isinserted between the HA tag and enzyme gene of the Aga2p fusionconstruct. Yeast display of sortases is induced upon the addition ofgalactose to the media.

FIG. 8. Synthesis of coenzyme A-conjugated sortase substrates. (A)Chemical structures of the SMCC crosslinker and coenzyme A (CoA). (B)Synthesis strategy for GGGK-CoA (SEQ ID NO: 35). (C) Synthesis strategyfor CoA-LPETGG (SEQ ID NO: 33). Also shown are the sequences of SEQ IDNOs: 35 and 50.

FIG. 9. Additional model screening results. (A) The indicated wt:C184AsrtA-yeast mixtures were modified with GGGK-CoA (SEQ ID NO: 35),incubated with 50 μM biotin-LPETGG (SEQ ID NO: 33) for 15 minutes, andsorted as described in FIG. 2. Analysis of the gene compositions beforeand after sorting by HindIII digestions reveals an enrichment factor of˜3,500-15,500 after a single round of sorting. (B) Yeast simultaneouslydisplaying the AviTag sequence and wild type E. coli biotin ligase(BirA) or its less active R317E mutant (Chapman-Smith A, Mulhern T D,Whelan F, Cronan J E, Jr., & Wallace J C (2001) The C-terminal domain ofbiotin protein ligase from E. coli is required for catalytic activity.Protein Science 10(12):2608-2617) were mixed in 1:1000 and 1:100BirA:R317E ratios. The mixtures were incubated with unmodifiedstreptavidin to silence the biotinylation signal that arises fromBirA-catalyzed biotinylation of the AviTag within the yeast secretorypathway during induction. The cells were treated with 1 μM biotin, 5 mMMgCl₂, and 0.2 mM ATP at room temperature for one hour. Followingstreptavidin-PE staining, the cells were subjected to FACS and the cellsthat exhibit the top 0.07% and 0.55% PE fluorescence intensities for the1:1000 and 1:100 screens, respectively, were collected. Analysis of genecompositions before and after sorting by HindIII digestions reveals anenrichment factor of ˜3,500-15,500 after one single round of sorting.

FIG. 10. FACS enables precise definition of sort gates using parallelcontrol samples. In this example, yeast cells displaying clone 4.2 weresubjected to identical reaction conditions and FACS analysis protocolsas the cells recovered after R6, enabling the creation of a sort gate(black polygon) that isolates mutants with higher specific activity thanclone 4.2 in the R7 sort. The percentage of cells residing within thesort gate is shown.

FIG. 11. Reaction conditions and sorting parameters used to evolvesortases with improved catalytic activity. Also shown are the sequencesof SEQ ID NOs: 33, 35, 36 and 37.

FIG. 12. The relative amount of biotinylated CoA adduct in thesupernatant is reflected by cell surface fluorescence afterSfp-catalyzed conjugation to yeast cells and streptavidin-PE staining.Biotin-CoA was mixed with GGGK-CoA (SEQ ID NO: 35) in various molarratios. A suspension of ICY200 cells at a density of 2.5×10⁷ cells/mLwas incubated with 6 μM Sfp and 5 μM total concentration of CoAconjugate. The fluorescence of the cells after streptavidin staining wasmeasured using flow cytometry.

FIG. 13. Sequences of clones isolated after (A) R7, (B) R8, (C) R9, and(D) R10mut.

FIG. 14. Representative kinetic measurements of the sortase-catalyzedreaction between Abz-LPETGK(Dnp)-CONH₂ (SEQ ID NO: 48) and GGG-COOH toyield Abz-LPETGGG (SEQ ID NO: 34). (A) Michaelis-Menten curves todetermine k_(cat) and K_(m LPETG) (SEQ ID NO: 32). (B) Michaelis-Mentencurves to determine K_(m GGG). For both (A) and (B), the overallreaction velocity is represented as turnovers per second (productconcentration/enzyme concentration). In every experiment, the enzymeconcentration was <1% of the substrate concentration and >1% of thesubstrate was converted to product, ensuring that multiple turnoverkinetics were measured.

FIG. 15. Comparison of the kinetic parameters of four evolved sortases.(A) Plots of reaction velocity (turnovers per second) vs. LPETG (SEQ IDNO: 32) peptide substrate concentration, with [GGG] fixed at 9 mM. (B)Plots of reaction velocity vs. GGG concentration, with [LPETG peptide](SEQ ID NO: 32) fixed at 1 mM. Due to its poor kinetics under the assayconditions, the plot for wt srtA is shown in the inset.

FIG. 16. Time course of turnovers by the evolvedP94R/D160N/D165A/K190E/K196T sortase. P94R/D160N/D165A/K190E/K196T srtA(914 pM) was incubated with 9 mM GGG and 1 mM Abz-LPETGK(Dnp) (SEQ IDNO: 48) substrate in 500 μL of reaction buffer. At 5-minute intervals,40-μL aliquots were removed, quenched, and analyzed by HPLC as describedin the Examples section. Each μM of product generated over the course ofthis experiment corresponds to approximately 1,092 turnover events.Averaged data and standard deviation from triplicate experiments areshown as open squares and bars, respectively. Fit lines were generatedby Mathematica according to the integrated Michaelis Menten equation,[Product]=[Substrate]₀−K_(m)ProductLog[Exp[([Substrate]₀−k_(cat)*time*[Enzyme])/K_(m)]*[Substrate]₀/K_(m)],where [Substrate]₀=1 mM and [Enzyme]=914 pM. The expected productconcentration from previously determined kinetic parameters is shown(black line) while a fit line to the data is shown (dashed). These datashow an r2 correlation of 0.983 with kinetic parameters k_(cat)=4.7±0.6s-1 and K_(m LPETG)=245±5 μM, compared with the parameters ofk_(cat)=5.4±0.4 s-1 and K_(m LPETG)=230±20 μM determined by endpointanalysis (Table 1, FIGS. 16, 17). The difference in observed k_(cat) isnot statistically significant by Students' t test to p>0.95.

FIG. 17. Cell-surface labeling with four evolved sortases. Live HeLacells expressing human CD154 conjugated at its extracellular C-terminusto LPETG (SEQ ID NO: 32) were incubated with 0.5 mM GGGYK-biotin (SEQ IDNO: 37) and no sortase A (no srtA) or 100 μM of the mutant sortase Ashown in the legend. The cells were stained withAlexaFluor594-conjugated streptavidin (SA-Alexa594) before flowcytometry analysis. Negative control reactions omitting sortase (green)or LPETG (SEQ ID NO: 32) (gray) are shown. Untreated cells stained withSA-594 (cyan) are also shown.

FIG. 18. Cell-surface reaction time courses to estimate substrateeffective molarity. Yeast displaying clones 4.2 and 4.3 were firstlabeled with GGGK-CoA (SEQ ID NO: 35) and then reacted with 1 μMbiotin-LPETGG (SEQ ID NO: 33) as described in the Examples section.Representative reaction progress curves for clone 4.2 (A) and 4.3 (B).The data was fit according to the equation described in the Examplessection. In this case, the 4.2 data show an r2 correlation of 0.999 witha cell surface GGG effective molarity estimate of 1.007 mM and a thetaestimate of 156 s, while the 4.3 data show an r2 correlation of 0.982with a GGG effective molarity estimate of 0.967 mM and a theta estimateof 0 s.

FIGS. 19-22. Reactivity profiles of a naïve library of SrtA mutants.Beginning from a library generated via random mutagenesis (as describedherein), the pentamutant Sortase A (P94R/D160N/D165A/K190E/K196T) wassubjected to four rounds of flow sorting under conditions of increasingstringency. In each round, biotinylated LPESG (SEQ ID NO: 38) wasreacted in the presence of at least a 10-fold excess of unbiotinylatedLPETG (SEQ ID NO: 32) and subjected to the selection described herein(left panel, each figure). Surviving library members were regrown andreinduced, then challenged with 10 μM Biotinyl-LPETG (SEQ ID NO: 32) andtheir reactivity measured (right panel, each figure). Over the course offour rounds of selection, virtually all LPETG (SEQ ID NO: 32) reactivitywas abolished, while competitive LPESG (SEQ ID NO: 38) reactivity roseto significant levels.

FIG. 23. The single clone identified from round 4 of the selectiondescribed in FIGS. 19-22 was subcloned and tested for activity on eitherLPETG (SEQ ID NO: 32) or LPESG (SEQ ID NO: 38) through an HPLC-basedsubstrate cleavage assay. Measuring the K_(cat) and K_(m) of the enzymefor each of these substrates, the novel Sortase 4S.4 was found to haveremarkably altered substrate specificity (>4000-fold, as determined byK_(cat)/K_(m)).

DEFINITIONS

The term “agent,” as used herein, refers to any molecule, entity, ormoiety. For example, an agent may be a protein, an amino acid, apeptide, a polynucleotide, a carbohydrate, a lipid, a detectable label,a binding agent, a tag, a metal atom, a contrast agent, a catalyst, anon-polypeptide polymer, a synthetic polymer, a recognition element, alinker, or chemical compound, such as a small molecule. In someembodiments, the agent is a binding agent, for example, a ligand, aligand-binding molecule, an antibody, or an antibody fragment.Additional agents suitable for use in embodiments of the presentinvention will be apparent to the skilled artisan. The invention is notlimited in this respect.

The term “amino acid,” as used herein, includes any naturally occurringand non-naturally occurring amino acid. Suitable natural and non-naturalamino acids will be apparent to the skilled artisan, and include, butare not limited to, those described in S. Hunt, The Non-Protein AminoAcids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C.Barrett, Chapman and Hall, 1985. Some non-limiting examples ofnon-natural amino acids are 4-hydroxyproline, desmosine,gamma-aminobutyric acid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and para-substituted phenylalanines (e.g., substituted with —C(═O)C₆H₅;—CF₃; —CN; -halo; —NO₂; —CH₃), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with —C(═O)C₆H₅; —CF₃; —CN; -halo;—NO₂; —CH₃), and statine. In the context of amino acid sequences, “X” or“Xaa” represents any amino acid residue, e.g., any naturally occurringand/or any non-naturally occurring amino acid residue.

The term “antibody,” as used herein, refers to a protein belonging tothe immunoglobulin superfamily. The terms antibody and immunoglobulinare used interchangeably. Antibodies from any mammalian species (e.g.,human, mouse, rat, goat, pig, horse, cattle, camel) and fromnon-mammalian species (e.g., from non-mammalian vertebrates, birds,reptiles, amphibia) are within the scope of the term. Suitableantibodies and antibody fragments for use in the context of someembodiments of the present invention include, for example, humanantibodies, humanized antibodies, domain antibodies, F(ab′), F(ab′)2,Fab, Fv, Fc, and Fd fragments, antibodies in which the Fc and/or FRand/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; and antibodies inwhich the FR and/or CDR1 and/or CDR2 regions have been replaced byhomologous human or non-human sequences. In some embodiments, so-calledsingle chain antibodies (e.g., ScFv), (single) domain antibodies, andother intracellular antibodies may be used in the context of the presentinvention. Domain antibodies, camelid and camelized antibodies andfragments thereof, for example, VHH domains, or nanobodies, such asthose described in patents and published patent applications of AblynxNV and Domantis are also encompassed in the term antibody. Further,chimeric antibodies, e.g., antibodies comprising two antigen-bindingdomains that bind to different antigens, are also suitable for use inthe context of some embodiments of the present invention.

The term “binding agent,” as used herein refers to any molecule thatbinds another molecule. In some embodiments, a binding agent bindsanother molecule with high affinity. In some embodiments, a bindingagent binds another molecule with high specificity. Examples for bindingagents include, without limitation, antibodies, antibody fragments,receptors, ligands, aptamers, and adnectins.

The term “bond-forming enzyme,” as used herein, refers to any enzymethat catalyzes a reaction resulting in the formation of a covalent bond.In some embodiments, the bond-forming enzyme is a sortase. In someembodiments, the bond-forming enzyme is a ligase, a polymerase, akinase, an aldolase, a diels alderase, or a transferase (e.g., abiotinyl transferase or a phosphopantathienyl transferase).

The term “conjugated” or “conjugation” refers to an association of twoentities, for example, of two molecules such as two proteins, or aprotein and a reactive handle, or a protein and an agent, e.g., adetectable label. The association can be, for example, via a direct orindirect (e.g., via a linker) covalent linkage or via non-covalentinteractions. In some embodiments, the association is covalent. In someembodiments, two molecules are conjugated via a linker connecting bothmolecules. For example, in some embodiments where two proteins areconjugated to each other to form a protein fusion, the two proteins maybe conjugated via a polypeptide linker, e.g., an amino acid sequenceconnecting the C-terminus of one protein to the N-terminus of the otherprotein. In some embodiments, conjugation of a protein to a protein orpeptide is achieved by transpeptidation using a sortase. See, e.g.,Ploegh et al., International PCT Patent Application, PCT/US2010/000274,filed Feb. 1, 2010, published as WO/2010/087994 on Aug. 5, 2010, andPloegh et al., International Patent Application PCT/US2011/033303, filedApr. 20, 2011, published as WO/2011/133704 on Oct. 27, 2011, the entirecontents of each of which are incorporated herein by reference, forexemplary sortases, proteins, recognition motifs, reagents, and methodsfor sortase-mediated transpeptidation.

The term “detectable label” refers to a moiety that has at least oneelement, isotope, or functional group incorporated into the moiety whichenables detection of the molecule, e.g., a protein or peptide, or otherentity, to which the label is attached. Labels can be directly attachedor can be attached via a linker. It will be appreciated that the labelmay be attached to or incorporated into a molecule, for example, aprotein, polypeptide, or other entity, at any position. In general, adetectable label can fall into any one (or more) of five classes: I) alabel which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁷⁶Br, ⁹⁹mTc (Tc-⁹⁹m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I,¹⁵³Gd, ¹⁶⁹Yb, and ¹⁸⁶Re; II) a label which contains an immune moiety,which may be antibodies or antigens, which may be bound to enzymes(e.g., such as horseradish peroxidase); III) a label which is a colored,luminescent, phosphorescent, or fluorescent moieties (e.g., such as thefluorescent label fluorescein-isothiocyanate (FITC); IV) a label whichhas one or more photo affinity moieties; and V) a label which is aligand for one or more known binding partners (e.g.,biotin-streptavidin, FK506-FKBP). In certain embodiments, a labelcomprises a radioactive isotope, preferably an isotope which emitsdetectable particles, such as β particles. In certain embodiments, thelabel comprises a fluorescent moiety. In certain embodiments, the labelis the fluorescent label fluorescein-isothiocyanate (FITC). In certainembodiments, the label comprises a ligand moiety with one or more knownbinding partners. In certain embodiments, the label comprises biotin. Insome embodiments, a label is a fluorescent polypeptide (e.g., GFP or aderivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., afirefly, Renilla, or Gaussia luciferase). It will be appreciated that,in certain embodiments, a label may react with a suitable substrate(e.g., a luciferin) to generate a detectable signal. Non-limitingexamples of fluorescent proteins include GFP and derivatives thereof,proteins comprising fluorophores that emit light of different colorssuch as red, yellow, and cyan fluorescent proteins. Exemplaryfluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP,mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2,EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO,mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry,mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See,e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein:properties, applications, and protocols Methods of biochemical analysis,v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, etal., Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP andnumerous other fluorescent or luminescent proteins. In some embodiments,a label comprises a dark quencher, e.g., a substance that absorbsexcitation energy from a fluorophore and dissipates the energy as heat.

The term “homologous”, as used herein is an art-understood term thatrefers to nucleic acids or polypeptides that are highly related at thelevel of nucleotide or amino acid sequence. Nucleic acids orpolypeptides that are homologous to each other are termed “homologues.”Homology between two sequences can be determined by sequence alignmentmethods known to those of skill in the art. In accordance with theinvention, two sequences are considered to be homologous if they are atleast about 50-60% identical, e.g., share identical residues (e.g.,amino acid residues) in at least about 50-60% of all residues comprisedin one or the other sequence, at least about 70% identical, at leastabout 80% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical,at least about 99.5% identical, or at least about 99.9% identical, forat least one stretch of at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 120, at least 150, or at least 200 amino acids.

The term “k_(cat)” refers to the turnover rate of an enzyme, e.g., thenumber of substrate molecules that the respective enzyme converts toproduct per time unit. Typically, k_(cat) designates the turnover of anenzyme working at maximum efficiency.

The term “K_(M)” is used herein interchangeably with the term “K_(m)”and refers to the Michaelis constant of an enzyme, an art-recognizedmeasure designating the substrate concentration at ½ the maximumreaction velocity of a reaction catalyzed by the respective enzyme.

The term “linker,” as used herein, refers to a chemical group ormolecule covalently linked to a molecule, for example, a protein, and achemical group or moiety, for example, a click chemistry handle. In someembodiments, the linker is positioned between, or flanked by, twogroups, molecules, or other moieties and connected to each one via acovalent bond, thus connecting the two. In some embodiments, the linkeris an amino acid or a plurality of amino acids (e.g., a peptide orprotein). In some embodiments, the linker is an organic molecule, group,polymer (e.g., PEG), or chemical moiety.

The term “mutation,” as used herein, refers to a substitution of aresidue within a sequence, e.g., a nucleic acid or amino acid sequence,with another residue, or a deletion or insertion of one or more residueswithin a sequence. Mutations are typically described herein byidentifying the original residue followed by the position of the residuewithin the sequence and by the identity of the newly substitutedresidue. For example, the term “P94S” in the context of describing amutation in the S. aureus sortase A protein describes a mutation inwhich the P (proline) residue at position 94 in the sortase A sequencehas been replaced by an S (serine) residue, the term “P94R” describes amutation in which the P (proline) residue at position 94 in the sortaseA sequence has been replaced by an R (arginine) residue, the term“E106G” describes a mutation in which the E (glutamate) residue atposition 106 in the sortase A sequence has been replaced by a G(glycine) residue, and so forth. See, e.g., SEQ ID NO: 1 for referenceof the respective amino acid residue positions in the wild type S.aureus sortase A protein.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof.

The term “reactive handle,” as used herein, refers to a reactive moietythat can partake in a bond-forming reaction under physiologicalconditions. Reactive handles can be used to conjugate entitiescomprising reactive handles that can react with each other to eachother. Examples of suitable reactive handles are, for example, chemicalmoieties that can partake in a click chemistry reaction (see, e.g., H.C. Kolb, M. G. Finn and K. B. Sharpless (2001). Click Chemistry: DiverseChemical Function from a Few Good Reactions. Angewandte ChemieInternational Edition 40 (11): 2004-2021). Some suitable reactivehandles are described herein and additional suitable reactive handleswill be apparent to those of skill in this art, as the present inventionis not limited in this respect.

The term “small molecule” is used herein to refer to molecules, whethernaturally-occurring or artificially created (e.g., via chemicalsynthesis) that have a relatively low molecular weight. Typically, asmall molecule is an organic compound (i.e., it contains carbon). Asmall molecule may contain multiple carbon-carbon bonds, stereocenters,and other functional groups (e.g., amines, hydroxyl, carbonyls, orheterocyclic rings). In some embodiments, small molecules are monomericand have a molecular weight of less than about 1500 g/mol. In certainembodiments, the molecular weight of the small molecule is less thanabout 1000 g/mol or less than about 500 g/mol. In certain embodiments,the small molecule is a drug, for example, a drug that has already beendeemed safe and effective for use in humans or animals by theappropriate governmental agency or regulatory body.

The term “sortase,” as used herein, refers to a protein having sortaseactivity, i.e., an enzyme able to carry out a transpeptidation reactionconjugating the C-terminus of a protein to the N-terminus of a proteinvia transamidation. The term includes full-length sortase proteins,e.g., full-length naturally occurring sortase proteins, fragments ofsuch sortase proteins that have sortase activity, modified (e.g.,mutated) variants or derivatives of such sortase proteins or fragmentsthereof, as well as proteins that are not derived from a naturallyoccurring sortase protein, but exhibit sortase activity. Those of skillin the art will readily be able to determine whether or not a givenprotein or protein fragment exhibits sortase activity, e.g., bycontacting the protein or protein fragment in question with a suitablesortase substrate under conditions allowing transpeptidation anddetermining whether the respective transpeptidation reaction product isformed. In some embodiments, a sortase is a protein comprising at least20 amino acid residues, at least 30 amino acid residues, at least 40amino acid residues, at least 50 amino acid residues, at least 60 aminoacid residues, at least 70 amino acid residues, at least 80 amino acidresidues, at least 90 amino acid residues, at least 100 amino acidresidues, at least 125 amino acid residues, at least 150 amino acidresidues, at least 175 amino acid residues, at least 200 amino acidresidues, or at least 250 amino acid residues. In some embodiments, asortase is a protein comprising less than 100 amino acid residues, lessthan 125 amino acid residues, less than 150 amino acid residues, lessthan 175 amino acid residues, less than 200 amino acid residues, or lessthan 250 amino acid residues.

Suitable sortases will be apparent to those of skill in the art andinclude, but are not limited to sortase A, sortase B, sortase C, andsortase D type sortases. Suitable sortases are described, for example,in Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclatureproposal for the various sortases of Gram-positive bacteria. ResMicrobiol. 156(3):289-97, 2005; Comfort D, Clubb R T. A comparativegenome analysis identifies distinct sorting pathways in gram-positivebacteria. Infect Immun., 72(5):2710-22, 2004; Chen I, Dorr B M, and LiuD R., A general strategy for the evolution of bond-forming enzymes usingyeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399; andPallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS inMicrobiology, 2001, 9(3), 97-101; the entire contents of each of whichare incorporated herein by reference). Any known sortase can be used asa starting enzyme in an evolution strategy provided herein, and theinvention is not limited in this respect. For example, the presentinvention encompasses embodiments relating to a sortase A from anybacterial species or strain. The invention encompasses embodimentsrelating to a sortase B from any bacterial species or strain. Theinvention encompasses embodiments relating to a class C sortase from anybacterial species or strain. The invention also encompasses embodimentsrelating to a class D sortase from any bacterial species or strain.Amino acid sequences of sortases and the nucleotide sequences thatencode them are known to those of skill in the art and are disclosed ina number of references cited herein, the entire contents of all of whichare incorporated herein by reference. Those of skill in the art willappreciate that any sortase and any sortase recognition motif can beused in some embodiments of this invention, including, but not limitedto, the sortases and sortase recognition motifs described in Ploegh etal., International PCT Patent Application, PCT/US2010/000274, filed Feb.1, 2010, published as WO/2010/087994 on Aug. 5, 2010; and Ploegh et al.,International Patent Application PCT/US2011/033303, filed Apr. 20, 2011,published as WO/2011/133704 on Oct. 27, 2011; the entire contents ofeach of which are incorporated herein by reference. The invention is notlimited in this respect.

In some embodiments, the sortase is sortase A of S. aureus. For example,in some embodiments, wild type sortase A from S. aureus serves as thestarting sortase, or parent sortase, for evolving an enhanced sortaseaccording to strategies and methods disclosed herein. The amino acidsequence of wild type sortase A of S. aureus is known to those of skillin the art, and a representative sequence(gi|21284177|ref|NP_(—)647265.1) is provided below:

(SEQ ID NO: 1) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.Additional S. aureus sortase A sequences will be apparent to those ofskill in the art and the invention is not limited in this respect. Insome embodiments, the sortase is a sortase A of another organism, forexample, from another bacterial strain, such as S. pyogenes. In someembodiments, the sortase is a sortase B, a sortase C, or a sortase D.Suitable sortases from other bacterial strains will be apparent to thoseof skill in the art, and the invention is not limited in this respect.

The term “sortase substrate,” as used herein refers to a molecule orentity that can be utilized in a sortase-mediated transpeptidationreaction. Typically, a sortase utilizes two substrates—a substratecomprising a C-terminal sortase recognition motif, and a secondsubstrate comprising an N-terminal sortase recognition motif and thetranspeptidation reaction results in a conjugation of both substratesvia a covalent bond. In some embodiments the C-terminal and N-terminalrecognition motif are comprised in the same protein, e.g., in the sameamino acid sequence. Sortase-mediated conjugation of the substrates insuch cases results in the formation of an intramolecular bond, e.g., acircularization of a single amino acid sequence, or, if multiplepolypeptides of a protein complex are involved, the formation of anintra-complex bond. In some embodiments, the C-terminal and N-terminalrecognition motifs are comprised in different amino acid sequences, forexample, in separate proteins. Some sortase recognition motifs aredescribed herein and additional suitable sortase recognition motifs arewell known to those of skill in the art. For example, sortase A of S.aureus recognizes and utilizes a C-terminal LPXT motif and an N-terminalGGG motif in transpeptidation reactions. Additional sortase recognitionmotifs will be apparent to those of skill in the art, and the inventionis not limited in this respect. A sortase substrate may compriseadditional moieties or entities apart from the peptidic sortaserecognition motif. For example, a sortase substrate may comprise an LPXTmotif, the N-terminus of which is conjugated to any agent, e.g., apeptide or protein, a small molecule, a binding agent, a lipid, acarbohydrate, or a detectable label. Similarly, a sortase substrate maycomprise a GGG motif, the C-terminus of which is conjugated to anyagent, e.g., a peptide or protein, a small molecule, a binding agent, alipid, a carbohydrate, or a detectable label. Accordingly, sortasesubstrates are not limited to proteins or peptides but include anymoiety or entity conjugated to a sortase recognition motif.

The term “target protein,” as used herein refers to a protein thatcomprises a sortase recognition motif. A target protein may be a wildtype protein, or may be an engineered protein, e.g., a recombinantprotein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Despite the many attractive features of enzymes as catalysts for organicsynthesis (1), as research tools (2-4), and as an important class ofhuman therapeutics (5, 6), the extent and diversity of theirapplications remain limited by the difficulty of finding in nature orcreating in the laboratory highly active proteins that catalyze chemicalreactions of interest. A significant fraction of protein catalystscurrently used for research and industrial applications was obtainedthrough the directed evolution of natural enzymes (7). Current methodsfor the directed evolution of enzymes have resulted in some remarkablesuccesses (8, 9) but generally suffer from limitations in reactionscope. For example, screening enzyme libraries in a multiwell format hasproven to be effective for enzymes that process chromogenic orfluorogenic substrates and is typically limited to library sizes ofapproximately 10²-10⁶ members, depending on the nature of the screen andon available infrastructure (10). Selections of cell-based librariesthat couple product formation with auxotrophy complementation (11) ortranscription of a reporter gene (12) enable larger library sizes to beprocessed but also suffer from limited generality because they rely onspecific properties of the substrate or product. Likewise, in vitrocompartmentalization is a powerful genotype-phenotype co-localizationplatform that has been used to evolve protein enzymes with improvedturnover but also requires corresponding screening or selection methodsthat thus far have been substrate- or product-specific (13).

Directed evolution strategies that are general for any bond-formingreaction would complement current methods that rely on screenablereactions or selectable properties of the substrate or product. Inprinciple, chemical complementation using an adapted yeast three-hybridassay is reaction-independent (14) but requires membrane-permeablesubstrates and offers limited control over reaction conditions becausethe bond-forming event must take place intracellularly. Phage-displayand mRNA-display systems that are general for any bond-forming reactionhave been used to evolve enzymes including DNA polymerases (15) and RNAligases (16). These approaches also offer advantages of larger librarysizes and significant control over reaction conditions because theenzymes are displayed extracellularly or expressed in the absence of ahost cell.

Some aspects of this invention relate to the recognition that cellsurface display (17-20) is an attractive alternative to phage and mRNAdisplay. In contrast to other display methods, the use of bacterial oryeast cells enables up to 100,000 copies of a library member to belinked to one copy of the gene, increasing sensitivity during screeningor selection steps. In addition, cell surface-displayed libraries arecompatible with powerful fluorescence-activated cell sorting (FACS) thatenable very large libraries to be screened efficiently (e.g., at ratesof >10⁷ cells per hour) with precise, quantitative control overscreening stringency. The multicolor capabilities of FACS also enablenormalization for enzyme display level during screening and simultaneouspositive and negative screens, capabilities that are difficult toimplement in phage and mRNA display.

Some aspects of this invention provide a technology that is based on anintegration of cell display (e.g., yeast display), enzyme-catalyzedsmall molecule-protein conjugation, and FACS into a general strategy forthe evolution of proteins that catalyze bond-forming reactions. Thetechnology was applied to evolve the bacterial transpeptidase sortase Afor improved catalytic activity, resulting in sortase variants with animprovement in activity of up to 140-fold. In contrast with wild type(WT) sortase, an evolved sortase enabled highly efficient cell-surfacelabeling of recombinant human CD 154 expressed on the surface of liveHeLa cells with a biotinylated peptide. The technology provided hereincan also be used to evolve other bond-forming enzymes, e.g., ligases,polymerases, kinases, transferases, aldolases, diels alderases, andtransferases (e.g., biotinyl transferases or phosphopantathienyltransferases), and additional bond-forming enzymes that can be evolvedusing the methods, reagents, and strategies disclosed herein will beapparent to those of skill in the art based on the instant disclosure.

Evolved Sortases with Enhanced Reaction Kinetics

Some aspects of this invention provide evolved sortases. In someembodiments, the evolved sortase exhibits an enhanced reaction kinetics,for example, in that it catalyzes a transpeptidation reaction at agreater speed or turnover rate than the respective wilt type sortase. Insome embodiments, the evolved sortase exhibits a modified substratepreference, for example, in that is utilizes a different substrate(e.g., a polypeptide comprising an altered sortase recognition motif) orbinds a given substrate with higher or lower affinity, or with higher orlower specificity than the respective wild type sortase. In someembodiments, the sortase recognizes a sortase recognition motif that therespective wild type sortase does not recognize or bind.

For example, some embodiments provide a sortase comprising an amino acidsequence that is homologous to the amino acid sequence of a wild typesortase (e.g., to the amino acid sequence of S. aureus Sortase A asprovided as SEQ ID NO: 1), or a fragment thereof. In some embodiments,the amino acid sequence of the provided sortase comprises one or moremutations as compared to the wild type sequence of the respectivesortase. For example, the evolved sortase sequence provided may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, or more mutations. In some embodiments, the sequence of the providedsortase is at least 90% identical, at least 95% identical, at least 98%identical, at least 99% identical, or at least 99.5% identical to a wildtype sortase sequence.

In some embodiments, an evolved S. aureus sortase A is provided. In someembodiments, the evolved sortase A comprises a mutation describedherein, for example, a P94S, P94R, E106G, F122Y, F154R, D160N, D165A,G174S, K190E, or K196T mutation, or a combination of any of thesemutations. In some embodiments, an evolved sortase is provided hereinthat comprises 1, 2, 3, 4, 5, 6, 7, 8, or all 9 of these mutations. Insome embodiments, an evolved sortase A is provided that comprises amutation that is homologous to the described mutations. For example, insome embodiments, an evolved sortase is provided that comprises a P94Sor P94R mutation, a D160N mutation, a D165A mutation, a K190E mutation,and a K196T mutation. In some embodiments, an evolved sortase isprovided that comprises a P94S or P94R mutation, a D160N mutation, and aK196T mutation. In some embodiments, an evolved sortase is provided thatcomprises a P94S or P94R mutation, a D160N mutation, and a D165Amutation. In some embodiments, an evolved sortase is provided thatcomprises a P94S or P94R mutation, a D160N mutation, a D165A mutation,and a K196T mutation.

Some evolved sortases provided herein exhibit enhanced reactionkinetics, for example, in that they can achieve a greater maximumturnover per time unit (k_(cat)) or a greater turnover per time atphysiological conditions. For example, in some embodiments, an evolvedsortase is provided herein that exhibits a k_(cat) that is at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least3.5-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or atleast 100-fold greater than the k_(cat) of the corresponding wild typesortase.

Some evolved sortases provided herein exhibit enhanced reactionspecificities, e.g., in that they bind a substrate with higher affinityor with higher selectivity, or in that they bind a substrate that is notbound or not efficiently bound by the respective wild type sortase. Forexample, some sortases provided herein exhibit a K_(M) for a substratebound by the corresponding wild type sortase that is at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, or at least 50-foldless than the K_(M) of the corresponding wild type sortase for thatsubstrate. Some evolved sortase A proteins provided herein, for example,exhibit a K_(M) for a substrate comprising a C-terminal sortaserecognition sequence of LPXT that is 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold less than the K_(M) of thecorresponding wild type sortase A for a substrate comprising aC-terminal sortase recognition sequence of LPXT.

In some embodiments, evolved sortases are provided that bind one oftheir substrates (e.g., a substrate with a C-terminal sortaserecognition motif) with a decreased K_(M) while exhibiting no or only aslight decrease in the K_(M) for another substrate (e.g., a substratewith an N-terminal sortase recognition motif). For example, some evolvedsortases provided herein exhibit a K_(M) for a substrate comprising aC-terminal sortase recognition motif (e.g., LPXT) that is at least2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or atleast 50-fold less than the K_(M) of the corresponding wild type sortasefor that substrate, and also exhibit a K_(M) for a substrate comprisingan N-terminal sortase recognition motif (e.g., GGG) that is not morethan 2-fold, not more than 5-fold, not more than 10-fold, or not morethan 20-fold greater than the K_(M) of the corresponding wild typesortase (e.g., wt S. aureus sortase A).

In some embodiments, evolved sortases are provided herein that exhibit aratio of K_(cat)/K_(M) for a substrate bound by the parent wild typesortase that is least 2-fold, at least 5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, at least 100-fold, or at least 120-foldgreater than the K_(cat)/K_(M) ratio of the corresponding wild typesortase.

Evolved Sortases with Altered Substrate Preferences

Some aspects of this invention provide evolved sortases that efficientlyuse substrates not bound by the respective parent wild type sortase. Forexample, in some embodiments, an evolved sortase is provided that isderived from a wild type S. aureus sortase A as the parent sortase A,which utilizes substrates comprising a C-terminal LPXT sortaserecognition motif and substrates comprising an N-terminal GGG sortaserecognition motif in a transpeptidation reaction. In some embodiments,the evolved sortases utilize a substrate different from those used bythe parent sortase, e.g., substrates comprising a C-terminal LPXS, LAXT,LAXTG (SEQ ID NO: 41), MPXT, MPXTG (SEQ ID NO: 42), LAXS, LAXSG (SEQ IDNO: 43), NPXT, NPXTG (SEQ ID NO: 44), NAXT, NAXTG (SEQ ID NO: 45), NAXS,NAXSG (SEQ ID NO: 46), LPXP, LPXPG (SEQ ID NO: 47), or LPXTA (SEQ ID NO:40) motif. In some embodiments, the evolved sortase comprises an S.aureus sortase A amino acid sequence, or a fragment thereof, with one ormore of the following mutations: P86L, N98S, A104T, A118T, F122S, D124G,N127S, K134R, K173E, K177E and 1182V.

Those of skill in the art will understand that the evolution technologyprovided herein allows for the generation of evolved sortasesrecognizing any desired recognition motif. For example, a desiredrecognition motif may be longer or shorter than the corresponding wildtype recognition motif, may comprise one or more amino acidsubstitutions, insertions, or deletions as compared to the correspondingwild type sortase recognition motif, or may be designed de novo, e.g.,not based on any naturally occurring sortase recognition motif. Theinvention is not limited in this respect.

Methods for Carrying Out Bond-Forming Reactions

Some aspects of this invention provide methods for carrying outbond-forming reactions, for example, sortase-mediated transpeptidationreactions using the evolved sortases described herein. In someembodiments, such methods comprise contacting an evolved sortaseprovided herein, or a sortase obtained by any of the evolution methodsdescribed herein, with a suitable substrate, e.g., a substratecomprising a suitable C-terminal sortase recognition motif and asubstrate comprising a suitable N-terminal sortase recognition motifunder conditions suitable for sortase-mediated transpeptidation. In someembodiments, the evolved sortase is a sortase A, for example, an evolvedS. aureus sortase A carrying one or more of the mutations describedherein. In some embodiments, the C-terminal sortase recognition motif isLPXT, e.g., LPETG (SEQ ID NO: 32), and/or the N-terminal recognitionmotif is GGG.

In some embodiments, at least one of the substrates is conjugated to asolid support. In some embodiments, at least one of the substrates isconjugated to the surface of a cell or other biological entity. Forexample, in some embodiments, at least one of the sortase substrates isexpressed as s fusion protein on the surface of a cell, e.g., a cellthat expresses a surface marker protein that is C-terminally fused to anamino acid sequence comprising a C-terminal sortase recognition motif(e.g., LPXT), or that is N-terminally fused to an N-terminal sortaserecognition motif (e.g., GGG).

The transpeptidation reactions provided herein typically result in thecreation of a protein fusion comprising the C-terminal sortaserecognition motif and the N-terminal sortase recognition motif. In someembodiments, one of the substrates (e.g., the substrate comprising theC-terminal sortase recognition motif) comprises a non-protein structure,e.g., a detectable label, a small molecule, a nucleic acid, a polymer,or a polysaccharide. It will be apparent to those of skill in the artthat the transpeptidation methods provided herein can be applied toconjugate any moieties that can be conjugated by any known sortase orsortase-mediated transpeptidation reaction, including, but not limitedto, the reactions and moieties disclosed in Ploegh et al., InternationalPCT Patent Application, PCT/US2010/000274, filed Feb. 1, 2010, publishedas WO/2010/087994 on Aug. 5, 2010; and Ploegh et al., InternationalPatent Application PCT/US2011/033303, filed Apr. 20, 2011, published asWO/2011/133704 on Oct. 27, 2011; the entire contents of each of whichare incorporated herein by reference, for exemplary sortases, proteins,recognition motifs, reagents, moieties, and methods for sortase-mediatedtranspeptidation. The invention is not limited in this respect.

Strategies for Directed Evolution of Bond-Forming Enzymes

Some aspects of this invention provide methods for the directedevolution of bond-forming enzymes. The evolution methods provided hereinare particularly suitable for the evolution of sortases, but, as will beapparent to the skilled artisan, they are not so limited. Anybond-forming enzyme can be evolved according to the strategies andmethods described herein. The methods described herein can be used,inter alia, to evolve bond-forming enzymes that exhibit enhancedreaction kinetics and/or altered substrate affinities or specificitiesas compared to the corresponding wild type enzyme.

In some embodiments, methods for the directed evolution of bond-formingenzymes are provided that involve providing a cell population in which afirst cell surface protein of a cell in the cell population isconjugated to a candidate bond-forming enzyme in a manner in whichdifferent cells within the population of cells comprise differentcandidate bond-forming enzymes conjugated to the cell surface protein.In some embodiments, the cells of the cell population also express asecond cell surface protein which is conjugated to a target substrate(substrate A). In some embodiments, the method comprises contacting thecell population with a second substrate (substrate B) conjugated to adetectable label under conditions suitable for the bond-forming enzymeto form a bond between the two substrates (A and B). Once the cells havebeen incubated for a period of time sufficient for a bond-forming enzymeto conjugate the substrates, any unconjugated substrate B can be washedaway, and cells expressing a bond-forming enzyme able to catalyze thedesired transpeptidation between A and B will retain the detectablelabel. These cells can be identified and/or isolated, and the identityof the expressed bond-forming enzyme can be determined.

The methods for evolving bond-forming enzymes provided herein can beused to evolve any enzyme, for example, sortases, ligases, polymerases,kinases, aldolases, diels alderases, and transferases (e.g., biotinyltransferases or phosphopantathienyl transferases), and additionalbond-forming enzymes that can be evolved using the methods, reagents,and strategies disclosed herein will be apparent to those of skill inthe art based on the instant disclosure. It will be apparent to those ofskill in the art that the choice of substrate A and/or B, of thedetectable label, and of identifying and/or isolating the cellsretaining the detectable label will depend on the enzyme to be evolved.For example, a sortase substrate will be used for the evolution ofsortases, a ligase substrate for the evolution of ligases, and so on.Reactions of enzymes that can directly add a detectable label to asubstrate, e.g., a transpeptidation reaction that adds a biotinylatedpeptide or a fluorescent protein to a target substrate, or a ligationreaction, polymerase reaction, or transferase reaction achieving similaradditions of a detectable label to a target substrate, can all be readout directly based on the respective cells retaining the detectablelabel. The evolution of enzymes that do not catalyze a reaction that candirectly add a detectable label to a target substrate, for example, ofkinases, which merely add a phosphate moiety to a substrate, may requirean alteration in the detection strategy. Rather than directly detectingproduct formation through the inclusion of a detectable label, e.g., abiotinylated tag, the reaction product can be detected indirectly insuch embodiments, for example, using an antibody raised against thereaction product (e.g., in the case of a kinase, the phosphorylatedproduct could be detected via an anti-phospho antibody). Direct andindirect labeling strategies are both suitable for downstream detectionmethods described herein or otherwise known in the art. For example, adirectly added biotin moiety can be detected via FACS afterstreptavidin-PE (phycoerythrin) staining, as described in more detail inthe Example section, while an indirectly detected reaction product maybe detected by an antibody that is conjugated to a detectable label,e.g., PE, biotin, or a fluorescent moiety.

In some embodiments, the evolution is based on cell display of thecandidate bond-forming enzyme in proximity to the target substrate. Insome embodiments, the cell display is bacterial display (using, e.g., E.coli or any other suitable bacterial strain). In some embodiments, thecell display is yeast display. For example, in some embodiments, themethod includes providing a yeast cell population in which a library ofcandidate bond-forming enzymes is expressed as a fusion to a surfaceprotein, e.g., an Aga2p cell surface mating factor. Preferably,different cells within the cell population express different candidatebond-forming enzymes. In some embodiments, a target substrate (substrateA) is also conjugated to a cell surface protein, e.g., an Aga1p cellsurface mating factor that is covalently bound to the Aga2p cell surfacemating factor. In some embodiments, the substrate is a peptide, and thepeptide is fused to the surface protein, e.g., to the N-terminus ofAgap1. The cell population displaying both the candidate bond-formingenzymes and the target substrate are then contacted with a secondsubstrate (substrate B) that is conjugated to a detectable label. Afterincubation under conditions suitable for the bond-forming enzyme to forma bond between substrate A and substrate B, unbound substrate B can bewashed away, while cells expressing a candidate bond-forming enzyme ableto conjugate substrate A and B will retain the detectable label. In someembodiments, labeled cells are detected and/or isolated. In someembodiments, the candidate bond-forming enzyme expressed in a cell thatretained the label is identified. Additional suitable display methodsand strategies will be apparent to those of skill in the art, and theinvention is not limited in this respect.

Some evolution methods provided herein include multiple rounds ofscreening a library of candidate bond-forming enzymes. In someembodiments, each round of screening is more stringent than the previousround for a desired characteristic of a bond-forming enzyme to beevolved. For example, in some embodiments, subsequent rounds ofevolution comprise decreasing concentrations of substrate B to selectfor bond-forming enzymes that can efficiently conjugate substrate A andB under conditions of low substrate B concentration. In someembodiments, bond-forming enzymes expressed in the cells isolated basedon the retention of the detectable label are subjected to adiversification procedure, for example, a random mutagenesis procedureor a DNA shuffling procedure (e.g., using staggered extension PCR(StEP), NeXT uracil excision recombination, or any other suitablemethod), thus creating a diversified library of candidate bond-formingenzymes, which can then be screened in a new round of selection forconjugation properties.

Methods and protocols for library diversification are well known tothose of skill in the art. In some embodiments, diversification mayinclude random mutation or recombination of isolated nucleic acidsequences encoding a parent bond-forming enzyme, and then expressing thediversified library as a fusion to a surface protein in a cellpopulation. In some embodiments, an evolution method provided hereinincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or more than30 cycles of library diversification and selection. In some embodiments,subsequent cycles may comprise a more stringent selection for a singleparameter (e.g., increased k_(cat)), or subsequent selections fordifferent parameters (e.g., select for altered substrate specificityfirst, and then for increased k_(cat) for the desired substrate). Theskilled artisan will understand that there is no limit to the number ofcycles of selection and library diversification, and that the methodsprovided herein can be used to achieve complex evolution processes withmultiple mutations affecting multiple enzyme characteristics. Theinvention is not limited in this respect.

The simultaneous display of bond-forming enzyme and target substrateincreases the effective concentration of these reaction partners, and,thus boosts screening efficiency. Conjugation of candidate bond-formingenzymes to surface proteins can be achieved by recombinant technologieswell known to those of skill in the art, e.g., expression of thecandidate enzyme as a fusion with a cell surface protein of therespective host cell, and expression of the target substrate as a fusionwith a second cell surface protein of the same host cell. Alternatively,either or both the candidate enzyme and the target substrate may beconjugated to a cell surface protein post-translationally, e.g., via areactive handle. Either or both the candidate enzyme and the targetsubstrate may be conjugated to the respective cell surface protein via alinker, and in some embodiments the linker may be cleavable, e.g., viaenzymatic or physical (e.g., UV light) cleavage.

Kits

Some aspects of this invention provide kits that comprise componentsuseful for performing a method for the directed evolution of abond-forming enzyme as described herein. In some embodiments, the kitcomprises an expression vector into which a library of candidatebond-forming enzymes can be cloned to be expressed as a fusion with acell surface protein, e.g., an Agap2 mating factor. In some embodiments,the kit also comprises reagents useful for screening a library ofbond-forming enzymes, e.g., a substrate conjugated to a detectablelabel. In some embodiments, cells for expression and/or screening of thelibrary of candidate bond-forming enzymes are also included.

Some aspects of this invention provide kits comprising an evolved,enhanced bond-forming enzyme (e.g., an evolved sortase as describedherein), and reagents useful for carrying out a bond-forming reactionusing the evolved enzyme. For example, in some embodiments, the kit maycomprise a nucleic acid encoding an amino acid sequence recognized bythe bond-forming enzyme, e.g., a C-terminal or N-terminal sortaserecognition motif, that can be used to generate protein fusions in whicha protein of interest carries a desired recognition motif. In someembodiments, an enzyme substrate conjugated to a detectable label isincluded.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the Examples below. Thefollowing Examples are intended to illustrate the benefits of thepresent invention and to describe particular embodiments, but are notintended to exemplify the full scope of the invention. Accordingly, itwill be understood that the Examples are not meant to limit the scope ofthe invention.

EXAMPLES Materials and Methods

Sortase Evolution.

A library of 7.8×10⁷ mutant sortase genes containing an average of 2.0amino acid changes per gene was introduced into yeast cells using gaprepair homologous recombination (see below for details on libraryconstruction). In Round 1, 6×10⁸ sortase library-expressing cells wereconjugated to GGGK-CoA (SEQ ID NO: 35), incubated with 100 μMbiotin-LPETGS (SEQ ID NO: 36) for 60 minutes, and stained withstreptavidin-PE and an AlexaFluor488-conjugated anti-HA antibody(Invitrogen). The top 1.4% of the PE/AlexaFluor488 double-positivepopulation were isolated and grown to saturation. At least a tenfoldexcess of cells relative to the number of cells recovered from sortingwere removed, pelleted, and induced to display enzymes at the cellsurface with galactose before entering the subsequent round of sorting.See FIG. 11 for details on screening stringency. Following round 4, thesurviving sortase genes were amplified by PCR and shuffled using theNeXT method (Muller K M, et al. (2005) Nucleotide exchange and excisiontechnology (NExT) DNA shuffling: a robust method for DNA fragmentationand directed evolution. Nucleic Acids Res 33(13):e117). The diversifiedgene library was introduced into yeast to generate a library of 6.9×10⁷transformants. Four additional rounds of enrichment were performed withGGG immobilized on the surface and biotinylated LPETG (SEQ ID NO: 32)peptide provided exogenously. For rounds 9, 9mut, and 10mut, the cellsfrom the previous round were modified with CoA-LPETGG (SEQ ID NO: 33) inTBS-B with 5 mM MgCl₂ and 5 mM CaCl₂ for 30 minutes to facilitateformation of the acyl-enzyme intermediate, before washing and initiatingthe reaction with 0.1-1.0 μM GGGYK-biotin (SEQ ID NO: 37).

Mammalian Cell Labeling.

HeLa cells were cultured at 37° C. in DMEM supplemented with 10% fetalbovine serum and 1% penicillin-streptomycin under an atmospherecontaining 5% CO₂. The cells were transfected with a 9:1 ratio ofplasmid pcDNA3-CD154-LPETG:cytoplasmic YFP expression plasmid (as atransfection marker) (SEQ ID NO: 32). After 24 hours, the transfectedcells were trypsinized, re-plated onto glass coverslips, and incubatedovernight at 37° C. Each coverslip was washed twice with Hank's balancedsalt solution (HBSS) and immersed into HBSS supplemented with 1 mMGGGYK-biotin (SEQ ID NO: 37), 5 mM CaCl₂, and 100 μM enzyme. After 5 to10 minutes, the coverslips were washed twice with PBS supplemented with1% bovine serum albumin (BSA), 1 mM unmodified GGG, and 5 mM MgSO4before immersion into a solution of streptavidin-AlexaFluor594 (1:200,Invitrogen) in PBS with 1% BSA and 5 mM MgSO4. For flow cytometryanalysis, the coverslips were washed twice with PBS before incubation inPBS on ice for 30 minutes. Cells were resuspended and analyzed using aBD Fortessa flow cytometer. The AlexaFluor594 fluorescence of the top16-25% most YFP-positive cells was recorded. For imaging, the coverslipswere washed twice with PBS containing 5 mM MgSO4 before analysis on aPerkin Elmer spinning disk confocal microscope (Harvard Center forBiological Imaging). Images were recorded using the DIC, YFP, and Alexachannels.

Methods for Sortase Reactions on Yeast Cells and Model Screens

Sortase Reactions on Yeast with Biotinylated GGG Peptide.

Saccharomyces cerevisiae cells displaying Staphylococcus aureus sortaseA and the S6 peptide (see below for details on induction of yeastdisplay) were resuspended to a cell density of 2.5×10⁸ cells/mL inTris-buffered saline (pH 7.5) with 1 mg/mL bovine serum albumin (TBS-B)and 5 mM MgCl₂ and incubated with 6 μM Sfp and 5 μM CoA-LPETGG (SEQ IDNO: 33) (see below for synthesis details) for 15 minutes. Cells werepelleted and washed with TBS-B before resuspension to a cell density of3×10⁶ to 1×10⁷ cells/mL in TBS-B with 5 mM CaCl₂ and 10 nM to 100 μMGGGYK-biotin peptide (SEQ ID NO: 37). After 15 to 60 minutes, thereactions were stopped by pelleting the cells and washing with ice-coldphosphate buffered saline with 1 mg/mL bovine serum albumin (PBS-B). Thecells were washed with ice-cold PBS-B containing 500 μM AAEK2(Astatech), an inhibitor of sortases (Maresso A W, et al. (2007)Activation of inhibitors by sortase triggers irreversible modificationof the active site. J Biol Chem 282(32):23129-23139), and 100 μMunmodified GGG (Sigma) before incubation with streptavidin-phycoerythrin(streptavidin-PE) (Fluka) and AlexaFluor488-conjugatedanti-hemagglutinin antibody (Invitrogen) to detect the extent of thesortase-catalyzed reaction and the enzyme display level, respectively.Cells were washed once more with PBS-B before flow cytometry analysis orFACS.

Sortase Reactions on Yeast with Biotinylated LPETG Peptide.

Yeast cells were conjugated to GGGK-CoA (SEQ ID NO: 35) (see below forsynthesis details) and reacted with the biotinylated LPETG (SEQ ID NO:32) peptide as described above. After stopping the reaction bycentrifuging and washing, the cells were resuspended in TBS-B containing5 μM TEV S219V protease (Kapust R B, et al. (2001) Tobacco etch virusprotease: mechanism of autolysis and rational design of stable mutantswith wild type catalytic proficiency. Protein Eng 14(12):993-1000) andincubated for 15-30 minutes to remove the background signal from theformation of any covalent acyl biotin-LPETG-enzyme (SEQ ID NO: 32)intermediate. After washing with cold PBS-B, the cells were stained withfluorophore-conjugated proteins as described above.

Model Screens.

Yeast displaying wild type sortase or the inactive C184A mutant weremixed in ratios of 1:1000 and 1:100 wt:C184A and treated as describedabove. After incubation with fluorophore conjugated proteins, 10⁷ cellsfrom each mixture were sorted in a MoFlo cell sorter (DakoCytomation).The top 0.06% and top 0.7% of the PE/AlexaFluor488 double positivepopulation for the 1:1000 and 1:100 experiments, respectively, werecollected. Collected cells were cultured until saturation in growthmedia (see below) with 50 μg/mL carbenicillin, 25 μg/mL kanamycin, and50 μg/mL streptomycin. Plasmid DNA was harvested using the Zymoprep kit(Zymo Research), and the recovered sortase genes were amplified usingthe primers 5′-CCCATAAACACACAGTATGTT (SEQ ID NO: 4) and5′-AATTGAAATATGGCAGGCAGC (SEQ ID NO: 12) and digested with HindIII todetermine the relative recovery of wild type and C184A genes.

Sortase Assay Methods

Flow Cytometry Activity Assay for Yeast Pools or Individual YeastClones.

A total of 1.25×10⁷ yeast cells were resuspended in 50 μL of TBS-Bcontaining 5 μM TEV S219V protease, 5 mM MgCl₂, and 5 mM CaCl₂. Afterincubation for 30 minutes at room temperature, the CoA-LPETGG (SEQ IDNO: 33) and GGGYK-biotin (SEQ ID NO: 37) peptides were added to the cellsuspension to final concentrations of 5 μM and 25 μM, respectively. Thecells were incubated at room temperature for an additional 30 minutesbefore Sfp was added to a final concentration of 6 μM. The cells wereincubated at room temperature for 7 minutes, pelleted by centrifugation,and washed with ice-cold PBS-B. The cells were stained withfluorophore-conjugated proteins as described above, washed, and analyzedby flow cytometry.

In Vitro Sortase Kinetics Assays.

See below for details on sortase expression and purification, and on thesynthesis of Abz-LPETGK(Dnp)-CONH₂ (SEQ ID NO: 48). Assays to determinek_(cat) and K_(m LPETG) were performed in 300 mM Tris pH 7.5, 150 mMNaCl, 5 mM CaCl₂, 5% v/v DMSO, and 9 mM Gly-Gly-Gly-COOH (GGG). Theconcentration of the LPETG (SEQ ID NO: 32) peptide substrate ranged from12.5 μM to 10 mM, and enzyme concentrations ranged from 25 nM to 1000nM. Assays for determination of K_(m GGG) were performed under the sameconditions, except the LPETG (SEQ ID NO: 32) peptide concentration wasfixed at 1 mM, the enzyme concentration was fixed at 41.5 nM, and theconcentration of GGG was varied from 33 μM to 30 mM, depending on theenzyme. Reactions were initiated with the addition of enzyme andincubated at 22.5° C. for 3 to 20 minutes before quenching with 0.5volumes of 1 M HCl. Five to ten nmol of peptide from the quenchedreactions were injected onto an analytical reverse-phase Eclipse XDB-C18HPLC column (4.6×150 mm, 5 μm, Agilent Technologies) and chromatographedusing a gradient of 10 to 65% acetonitrile with 0.1% TFA in 0.1% aqueousTFA over 13 minutes. Retention times under these conditions for theAbz-LPETGK(Dnp)-CONH₂ (SEQ ID NO: 48) substrate, the released GKDnppeptide, and the Abz-LPETGGG-COOH (SEQ ID NO: 34) product were 12.8,10.4, and 9.1 min, respectively. To calculate the percent conversion,the ratio of the integrated areas of the Abz-LPETGGG-COOH (SEQ ID NO:34) and Abz-LPETGK(Dnp)-CONH₂ (SEQ ID NO: 48) peptide Abs220 peaks wascompared to a standard curve generated by mixing the product andstarting peptide in known ratios. To determine k_(cat) and K_(m),reaction rates were fit to the Michaelis-Menten equation using OriginPro7.0 software. All kinetics values reported represent the average of atleast three measurements.

Substrate Synthesis Methods

Biotin-LC-LELPETGG-CONH₂ (SEQ ID NO: 49), Fmoc-GGGK-CONH₂ (SEQ ID NO:35), and NH₂—YLELPETGG-CONH₂ (SEQ ID NO: 50) were purchased fromGenscript and used without further purification. NH₂-GGGYK(biotin)-CONH₂(SEQ ID NO: 37) was purchased from Genscript and purified usingreverse-phase HPLC on a C18 column. Biotin-LCYGLPETGS-CONH₂ (SEQ ID NO:52) was purchased from New England Peptide and used without furtherpurification.

Synthesis of GGGK-CoA (SEQ ID NO: 35).

Fmoc-GGGK-CONH₂ (SEQ ID NO: 35) was dissolved in DMSO to a finalconcentration of 100 mM, and 1.5 equivalents of sulfo-SMCC(Thermo-Fisher) and 2 equivalents of DIPEA (Sigma) in DMSO were added.The reaction was incubated for 1 hr at room temperature, then added to1.5 equivalents of coenzyme A trilithium hydrate (Sigma) in DMSO to afinal peptide concentration of 25 mM and mixed at room temperatureovernight. If appropriate, the Fmoc protecting group was removed with20% vol/vol piperidine and incubation for 20 minutes. The reaction wasquenched by the addition of 1 equivalent of TFA, and the product waspurified on a preparative Kromasil 100-5-C18 column (21.2 ˜250 mm, PeekeScientific) by reverse phase HPLC (flow rate: 9.5 mL/min; gradient: 10%to 70% acetonitrile with 0.1% TFA in 0.1% aqueous TFA gradient over 30minutes; retention time: 17.1 minutes). ESI-MS (found):[M-H]−m/z=1300.1. Calculated for C45H72N14O23P3S—: m/z=1301.4. Theconcentration of GGGK-CoA (SEQ ID NO: 35) peptide was determined fromthe measured A259 using the known molar extinction coefficient ofcoenzyme A, 15,000 M⁻¹ cm⁻¹ (Killenberg P G & Dukes D F (1976) CoenzymeA derivatives of bile acids-chemical synthesis, purification, andutilization in enzymic preparation of taurine conjugates. J Lipid Res17(5):451-455).

Synthesis of CoA-LPETGG

(SEQ ID NO: 33) NH₂—YLELPETGG-CONH₂ (SEQ ID NO: 50) (0.0084 mmol) wasincubated with sulfo-SMCC (0.021 mmol, 2.5 eq.) in 142 μL of DMSO and 3μL DIPEA (0.017 mmol, 2.0 equivalents) for 2 hours at room temperature.The maleimide adduct was purified using reverse-phase HPLC on apreparative C18 column (flow rate: 9.5 mL/min; gradient: 10% to 60%acetonitrile with 0.1% TFA in 0.1% aqueous TFA over 30 minutes;retention time: 22.0 minutes). After lyophilization of the collectedpeak, the white solid was dissolved in 0.1 M phosphate buffer pH 7.0with 45% acetonitrile. Coenzyme A trilithium hydrate (11.2 mg) wasadded, and the reaction was incubated at one hour at room temperature.The desired product was obtained after purification on a C18 column(flow rate: 9.5 mL/min flow rate; 0% to 50% acetonitrile in 0.1 Mtriethylammonium acetate over 30 minutes; retention time: 21.9 minutes).ESI-MS (found): [M-H]−m/z=1961.8. Calculated for C₇₇H₁₁₆N₁₈O₃₄P₃S—:m/z=1961.7. The concentration of CoA-LPETGG (SEQ ID NO: 33) peptide wasdetermined as described above for GGGK-CoA (SEQ ID NO: 35).

Abz-LPETGK(Dnp)-CONH₂ Substrate for HPLC Assays (SEQ ID NO:48).

This compound was synthesized at 200 μmol scale using an AppliedBiosystems 433A peptide synthesizer. 200 μmol-equivalents of NovaPEGRink Amide resin (EMD biosciences) were loaded onto the machine andcoupled using 5 equivalents of each Fmoc-protected amino acid buildingblock with standard acid labile side-chain protecting groups (Thr(OtBu),Glu(OtBu)) and using Fmoc Lysine(Dnp) (Chem-Impex). Terminal couplingwith Boc 2-Aminobenzoic Acid (Chem-Impex) yielded the fully protectedpeptide, which was cleaved by three 1-hour treatments with 20 mL of 95%TFA+2.5% water+2.5% triisopropylsilane (Sigma). The cleavage mixtureswere pooled and concentrated by rotary evaporation, and the peptide wasprecipitated by the addition of 9 volumes of ice-cold diethyl ether. Thesamples were purified by reverse phase HPLC as described above forGGGK-CoA (SEQ ID NO: 35) (retention time: 28 minutes), pooled andconcentrated by lyophilization. The concentration of the peptide wasdetermined by the known molar extinction coefficient of the Dnp group,fĀ355 nm=17,400 M-1 cm-1 (Carsten M E & Eisen H N (1953) The Interactionof Dinitrobenzene Derivatives with Bovine Serum Albumin. Journal of theAmerican Chemical Society 75(18):4451-4456).

Cloning Methods Including Library Generation

Primers SEQ Primer Sequence ID 1FTCCAGACTATGCAGGATCTGAGAACTTGTACTTTCAAGGTGC  2 TAGCCAAGCTAAACCTCA 1RCAGAAATAAGCTTTTGTTCGGATCCTTTGACTTCTGTAGCTACAAAG  3 2FCCCATAAACACACAGTATGTT  4 2RACCTTGAAAGTACAAGTTCTCAGATCCTGCATAGTCTGGAACGTCGT  5 3FAAAGATAAACAATTAACATTAATTACTGCTGATGATTACAATGAA  6 3RATCTCGAGCTATTACAAGTCCTCTTCAGAAATAAGCTTTTGTTCGGA  7 4FGTGGAGGAGGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGT  8 4RAGTAATTAATGTTAATTGTTTATCTTT  9 5F TGGGAATTCCATATGCAAGCTAAACCTCAAATTCCG10 5R TTTTTTCTCGAGTTTGACTTCTGTAGCTACAAAG 11 6R AATTGAAATATGGCAGGCAGC 127F CCAGGACCAGCAACAAGYGAACAATTAAATAGA 13 8FATGACAAGTATAAGAAAYGTTAAGCCAACAGCKGTAGAAGTTCTAGAT 14 9FTTAATTACTTGTGATGGKTACAATGAAAAGACA 15 Y = C, T; K = G, T; underlinednucleotides represent mixtures of 70% the indicated nucleotide and 10%each of the remaining three nucleotides

YIPlac211-GPD-S6-Aga1p and Integration Into the Yeast Genome. TheYIPlac211-GPD-Avitag-Aga1p plasmid, constructed by ligation of theAvitag-Aga1p gene into YIPlac211 (ATCC) at BamHI/SacI and ligation ofthe glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter sequence atXbaI/BamHI, served as the starting point. The S6 peptide sequence wasinserted after the signal sequence and before 11e30 of Aga1p by overlapextension PCR. The extended PCR product was digested with BamHI andBsiWI and ligated into similarly digested YIPlac211-GPD-Avitag-Aga1pplasmid, resulting in the yeast integrating plasmidYIPlac211-GPDS6-Aga1p.

To integrate the plasmid into the genome, YIPlac211-GPD-S6-Aga1p waslinearized by digestion with BsiWI and transformed into S. cerevisiaestrain BJ5465 with lithium acetate, selecting for transformantsharboring the integrated plasmid on solid media lacking uracil. A yeastcolony with the S6-Aga1p construct correctly inserted was designatedICY200 and displays the S6 peptide sequence constitutively on the cellsurface as a fusion to the N-terminus of Aga1p.

pCTCon2CTEV-wt srtA and pCTCon2CTEV-srtA C184A (.HindIII).

The pCTCon2CTEV-wt srtA plasmid was constructed inside yeast through athree-part, gap repair homologous recombination process (Raymond C K,Pownder T A, & Sexson S L (1999) General method for plasmid constructionusing homologous recombination. BioTechniques 26(1):134-138, 140-131).The pCTCon2B-BirA plasmid, which was constructed from pCTCon2 andexpresses the Aga2p-linker-HA-E. coli biotin ligase-myc construct,served as the starting point. S. aureus genomic DNA was amplified withprimers 1F and 1R, and pCTCon2B-BirA with primers 2F and 2R. These twoproducts were transformed together with PstI/BamHI-digestedpCTCon2B-BirA into S. cerevisiae strain ICY200 to yield thepCTCon2CTEV-wt srtA plasmid. The cloned sortase A gene lacks theN-terminal 59 amino acids, which do not impact catalytic activity(Ilangovan U, Ton-That H, Iwahara J, Schneewind O, & Clubb R T (2001)Structure of sortase, the transpeptidase that anchors proteins to thecell wall of Staphylococcus aureus. Proc Natl Acad Sci USA98(11):6056-606), but these amino acids are still included in thenumbering for the mutations.

To introduce the C184A mutation, pCTCon2CTEV-wt srtA was separatelyamplified with primer pairs 3F/3R and 4F/4R, and the two gene fragmentswere transformed into ICY200 together with NheI/BamHI-digestedpCTCon2CTEV-wt srtA. The HindIII site within the myc coding sequence wasthen removed using an analogous process, allowing the wt and C184Aplasmids to be distinguished by a HindIII restriction digest.

pET29 Sortase Expression Plasmids

Sortase genes were subcloned into pET29 at NdeI and XhoI using theprimers 5F and 5R. Plasmids encoding sortase single mutants wereconstructed using the Quikchange method. All expressed sortases lack theN-terminal 59 amino acids.

Sortase A Library R0.

The round zero (R0) sortase A library was cloned into S. cerevisiaeICY200 using gap repair homologous recombination. The wild type sortaseA gene, lacking the N-terminal 59 amino acids, was mutagenized in PCRreactions containing 5 μM 8-oxo-2 fdeoxyguanosine (8-oxo-dGTP), 5 μM6-(2-deoxy-b-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][1,2]oxazin-7-one(dPTP), 200 μM each dNTP, and 0.4 μM each of primers 1F and 1R.Reactions were thermocycled ten times and the mutagenized genes werefurther amplified in PCR reactions without mutagenic dNTP analogs usingprimers 1F and 3R. Gel-purified genes and NheI/BamHI-digestedpCTCon2CTEV-wt srtA were combined in a 1:3 mass ratio, concentrated byethanol precipitation, and electroporated into competent ICY200 asdescribed, resulting in a library of 7.8×10⁷ transformants. A total of˜10⁹ cells from the fully grown library culture were pelleted andinduced as described below.

Recombined Sortase A Library (R4shuf).

In vitro recombination was performed using the NExT procedure (1).Sortase genes recovered after R4 were amplified with the primers 2F and6R in PCR reactions containing 50 μM dUTP, 150 μM dTTP, and 200 μM eachof dATP, dCTP, and dGTP. After purification by gel extraction, 17 μg ofthe PCR product was incubated with 7.5 units of uracil deglycosylase(NEB). Piperidine was added (10% vol/vol) and the reaction was heated at90° C. for 3 min. The resulting gene fragments were purified using theQiaExII kit after neutralization of the piperidine with glacial aceticacid.

Fragments were assembled in PCR reactions containing 1 μg of fragmentsusing the conditions reported by Tawfik (Herman A & Tawfik D S (2007)Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): aversatile tool for generating targeted libraries. Protein Eng Des Sel20(5):219-226). In separate fragment assembly reactions, primers 7F, 8F,and 9F were each added to the assembly reaction at 0.5 μM and 1.5 μM tofavor the inclusion of P94S, D160N, D165A, and D186G. mutations thatappeared to improve catalytic efficiency based on activity assays ofindividual clones evolved in R3 and R4. Assembly reactions were purifiedusing the Qiaquick kit, reamplified with the primers 1F and 1R, andpurified by gel extraction. Eight μg of each assembled gene product (24μg total) were mixed with 7 μg of NheI/BamHI-digested pCTCon2CTEVplasmid, concentrated by ethanol precipitation, and electroporated intocompetent ICY200 cells as described above, resulting in a library of6.9×10⁷ transformants. A total of ˜10⁹ cells from the fully grownlibrary culture were pelleted and induced as described below.

Sortase Library R8mut.

The R8mut library was cloned into yeast as described above for the R0library, starting with an equimolar mixture of the genes encoding clones8.3, 8.4, 8.5, and 8.9 (see FIG. 13). These clones were chosen becausethey possessed only one extraneous mutation in addition to thetetramutant motif (8.3, 8.4, 8.9), or because they possessed an alteredtetramutant core (8.5). More heavily mutagenized library members (8.11,8.13) were avoided in order to minimize deviation from the tetramutantcore of mutations. The concentrations of dPTP and 8-oxo-dGTP were each10 μM. Following electroporation into ICY200 as described above, alibrary of 5×10⁷ transformants was obtained with a bulk mutagenesis rateof 1.5%, corresponding with an amino acid mutagenesis rate of 1.1%.

General Yeast Methods

Yeast cells were transformed with DNA using the lithium acetate method.Plasmid DNA from yeast cultures was harvested using the Zymoprep YeastPlasmid Minipreparation Kit (Zymo Research) following the manufacturer'sinstructions. For sequencing, zymoprepped DNA was amplified bytransformation into E. coli, or sortase genes were amplified by PCRusing the primers 2F and 6R.

S. cerevisiae strain ICY200 was propagated in YPD or growth mediaconsisting of 100 mM phosphate pH 6.6, 2% (w/v) dextrose, 0.67% yeastnitrogen base (Sigma), 100 μg/mL cysteine, 100 μg/mL proline, 30 μg/mLhistidine, 30 μg/mL methionine, and complete supplement mixture lackinguracil (MP Biomedical). Growth media for ICY200 transformed with thepCTCon2CTEV yeast display plasmids was the same, except the completesupplement mixture lacked uracil and tryptophan. Induction media was thesame as the growth media lacking uracil and tryptophan, except thecarbon source consisted of 1.8% galactose and 0.2% dextrose. Forinduction of display of sortases on the cell surface, yeast cells from afully grown culture were pelleted, resuspended in induction media at adensity of 7×10⁶ cells/mL, and incubated at 20° C. for 18-36 hours.Cells were pelleted and washed with TBS supplemented with 1 mg/mL BSA(TBS-B) before input into assays.

Protein Expression and Purification

Bacterial Expression of Sortases.

E. coli BL21(DE3) transformed with pET29 sortase expression plasmidswere cultured at 37° C. in LB with 50 μg/mL kanamycin untilOD600=0.5-0.8. IPTG was added to a final concentration of 0.4 mM andprotein expression was induced for three hours at 30° C. The cells wereharvested by centrifugation and resuspended in lysis buffer (50 mM TrispH 8.0, 300 mM NaCl supplemented with 1 mM MgCl₂, 2 units/mL DNAseI(NEB), 260 nM aprotinin, 1.2 μM leupeptin, and 1 mM PMSF). Cells werelysed by sonication and the clarified supernatant was purified on Ni-NTAagarose following the manufacturer's instructions. Fractions thatwere >95% purity, as judged by SDS-PAGE, were consolidated and dialyzedagainst Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl). Enzymeconcentration was calculated from the measured A280 using the publishedextinction coefficient of 17,420 M-1 cm-1 (Kruger R G, et al. (2004)Analysis of the substrate specificity of the Staphylococcus aureussortase transpeptidase SrtA. Biochemistry 43(6):1541-1551).

Bacterial Expression of Sfp Phosphopantetheinyl Transferase.

E. coli BL21(DE3) harboring the pET29 expression plasmid for Sfpphosphopantetheinyl transferase (a gift from the Christopher T. Walshlab) were cultured at 37° C. in LB with 50 μg/mL kanamycin until OD600˜0.6. IPTG was added to a final concentration of 1 mM, and proteinexpression was induced at 37° C. for three hours. The cells wereharvested by centrifugation and lysed by resuspension in B-PER (Novagen)containing 260 nM aprotinin, 1.2 μM leupeptin, 2 units/mL DNAseI, and 1mM PMSF. The clarified supernatant was purified on Ni-NTA agarose, andfractions that were >95% pure were consolidated and dialyzed against 10mM Tris pH 7.5+1 mM EDTA+5% glycerol. Enzyme concentration wascalculated from the measured A280 using the published extinctioncoefficient of 27,220 M⁻¹ cm⁻¹ (Mofid M R, Finking R, Essen L O, &Marahiel M A (2004) Structure-based mutational analysis of the4′-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrierprotein recognition and reaction mechanism. Biochemistry43(14):4128-4136).

Bacterial Expression of TEV S219V Protease.

E. coli BL21(DE3) harboring the pRK793 plasmid for TEV S219V expressionand the pRIL plasmid (Addgene) were cultured in LB with 50 μg/mLcarbenicillin and 30 μg/mL chloramphenicol until OD600 ˜0.7. IPTG wasadded to a final concentration of 1 mM, and the cells were induced forthree hours at 30° C. The cells were pelleted by centrifugation andlysed by sonication as described above for the sortases. The clarifiedlysate was purified on Ni-NTA agarose, and fractions that were >95% TEVS219V were consolidated and dialyzed against TBS. Enzyme concentrationwas calculated from the measured A280 using the reported extinctioncoefficient of 32,290 M-1 cm-1 (Tropea J E, Chemy S, & Waugh D S (2009)Expression and purification of soluble His(6)-tagged TEV protease.Methods Mol Biol 498:297-307).

Protein Sequences

Amino acid changes relative to wild type S. aureus sortase A areunderlined. Some sequences are displayed with C-terminal 6×His tag.Sequences without the 6×His tag are also functional and are provided bythe sequences below as well.

Aga2p-srtA Cl 84A (FIG. 2, FIG. 3B) (SEQ ID NO: 16)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTLQASGGGGSGGGGSGGGGSYPYDVPDYAGSENLYFQGASQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKSKDKQLTLITADDYNEKTGVWEKRKIFVATEVKGSEQKLISEEDLAga2p-Clone 8.3 (FIG. 3B, FIG. 4A) (SEQ ID NO: 17)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTLQASGGGGSGGGGSGGGGSYPYDVPDYAGSENLYFQGASQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKSKDKQLTLITCDDYNEKTGVWETRKIFVATEVKGSEQKLISEEDLAga2p-Clone 8.4 (FIG. 3B, FIG. 4A) (SEQ ID NO: 18)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTLQASGGGGSGGGGSGGGGSYPYDVPDYAGSENLYFQGASQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKSKDKQLTLITCDDYNEETGVWETRKIFVATEVKGSEQKLISEEDLAga2p-Clone 8.9 (FIG. 3B, FIG. 4A) (SEQ ID NO: 19)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTLQASGGGGSGGGGSGGGGSYPYDVPDYAGSENLYFQGASQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYRMTSIRNVKPTAVEVLDEQKSKDKQLTLITCDDYNEKTGVWETRKIFVATEVKGSEQKLISEEDLAga2p-Clone 8.13 (FIG. 3B, FIG. 4A) (SEQ ID NO: 20)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTLQASGGGGSGGGGSGGGGSYPYDVPDYAGSENLYFQGASQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEGNESLDDQNISIAGHTYIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKSKDKQLTLITCDDYNEKTGVWETRKIFVATEVKGSEQKLISEEDLwild type S. aureus sortase A (FIG. 3B, FIG. 4A, Table 1)(SEQ ID NO: 21)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH srtA P94S (Table 1)(SEQ ID NO: 22)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH srtA D160N (Table 1)(SEQ ID NO: 23)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH srtA D165A (Table 1)(SEQ ID NO: 24)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTAVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH srtA K196T (Table 1)(SEQ ID NO: 25)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIFVATEVKLEHHHHHHClone 4.2 (FIG. 3B, FIG. 4A, Table 1) (SEQ ID NO: 26)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTDVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLEHHHHHHClone 4.3 (FIG. 3B, FIG. 4A, Table 1) (SEQ ID NO: 27)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTAVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH P94S/D160N/D165A/K196T (Table 1)(SEQ ID NO: 28)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIFVATEVKLEHHHHHHP94S/D160N/K196T (Table 1, FIG. 5) (SEQ ID NO: 29)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIFVATEVKLEHHHHHH P94S/D160N/D165A (Table 1)(SEQ ID NO: 30)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHHP94R/D160N/D165A/K190E/K196T (Table 1) (SEQ ID NO: 31)MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLEHHHHHHEffective Molarity of Surface-Conjugated Substrate Relative toYeast-Displayed Sortase

Yeast displaying clones 4.2 and 4.3 were conjugated with GGGK-CoA (SEQID NO: 35) as described in the Materials and Methods section. Theresulting cells were incubated with 1 μM Biotin-LPETGG (SEQ ID NO: 33)in TBS with 5 mM CaCl₂, and aliquots were removed at various time pointsand immediately diluted 1:20 into ice-cold PBS containing 6 μM TEVS219V, 5 mM AAEK2 (an inhibitor of S. aureus sortase A (2)), 1 mMberberine chloride (an inhibitor of S. aureus sortase A (Kim S H, et al.(2004) Inhibition of the bacterial surface protein anchoringtranspeptidase sortase by isoquinoline alkaloids. Biosci BiotechnolBiochem 68(2):421-424)), and 5 mM of non-biotinylated GGG.

After incubation on ice for fifteen minutes, the samples were pelletedand resuspended in ice-cold PBS containing 6 μM TEV S219V, 5 mM AAEK2, 5mM GGG for one hour. Following staining of the cells withstreptavidin-phycoerythrin (for reaction extent) andAlexaFluor488-conjugated antihemagglutinin antibody (for display), thephycoerythrin mean fluorescence intensity (PE MFI) of theAlexaFluor488-positive cells were recorded with a BD Fortessa flowcytometer. When plotted versus time (t), the PE MFI (for reactionextent) for the 488/PE-double positive population of each sample werefit to the Poisson equation representing the proportion of sitesconverted for a reaction operating at constant velocity v,f_(∞)(1−e^(−v)*^((t+theta)))+f₀. The scaling factor f_(∞) is taken torepresent the fluorescence intensity of a fully labeled cell and isdetermined by allowing reactions to run for two hours and fixing this asthe endpoint. The minimum fluorescence intensity for a library member,f₀, is fixed from the PE MFI of 488-negative cells within thepopulation. The velocity of the reaction, v, and the time correctionfactor, theta, were both determined by nonlinear regression of the datato the fit curve using the program Mathematica. The velocity data werethen transformed into estimates for the effective molarity of displayedenzymes for [GGG] by the use of the previously determinedMichaelis-Menten relations for clones 4.2 and 4.3,[GGG]=K_(m,GGG)*K_(m,LPETG)*v+K_(m,GGG)*[LPETG]*v/(k_(cat)*[LPETG]−K_(m,LPETG)*v−[LPETG]*v)[GGG] estimates were made for two technical replicates each of the 4.2and 4.3 sortase mutants, and the overall estimate of [GGG] was found tobe 0.95±0.11 mM; the LPETG sequence of the equation corresponds to SEQID NO: 32).

Results

The evolution strategy provided herein was validated using model screensfor Staphylococcus aureus sortase A-catalyzed transpeptidation activity,resulting in enrichment factors of 6,000-fold after a single round ofscreening. The strategy provided herein was applied to evolve sortase Afor improved catalytic activity. After eight rounds of screening, weisolated variants of sortase A with up to a 140-fold increase inLPETG-coupling (SEQ ID NO: 32) activity compared with the starting wildtype enzyme. An evolved sortase variant enabled much more efficientlabeling of LPETG-tagged (SEQ ID NO: 32) human CD154 expressed on thesurface of HeLa cells compared with wild type sortase.

Design and Implementation of a General System for the Evolution ofBond-Forming Enzymes.

The enzyme evolution system is overviewed in FIG. 1. Yeast cells displaythe enzyme library extracellularly as a fusion to the Aga2p cell surfacemating factor, which is covalently bound to the Aga1p mating factor witha reactive handle that enables covalent attachment of substrate A tocells. We chose the S6 peptide (3) as the reactive handle to linksubstrate A to cells using Sfp phosphopantetheinyl transferase fromBacillus subtilis. Substrate B linked to an affinity handle (e.g.,biotin, represented by the gray circle in FIG. 1) is added to thesubstrate A-conjugated yeast display enzyme library. Because of the higheffective molarity of substrate A with respect to each cell's displayedlibrary member, both of which are immobilized on the cell surface,active library members will predominantly catalyze thepseudointramolecular A-B bond formation between affinity handle-linkedsubstrate B and substrate A molecules on their own host cell. Theintermolecular coupling of substrate B with substrate A moleculesattached to other cells is entropically much less favorable, andtherefore yeast cells displaying inactive enzymes should remainpredominantly uncoupled to the affinity handle.

Following incubation with substrate B for the desired reaction time,cells are stained with a fluorescent molecule that binds the affinityhandle [e.g., streptavidin-phycoerythrin (streptavidin-PE)]. The mostfluorescent cells, which encode the most active catalysts, are isolatedby FACS. Up to 10⁸ cells can be sorted in a 2-h period using modern FACSequipment. After sorting and growth amplification, the recovered cellscan be enriched through additional FACS steps, or DNA encoding activelibrary members can be harvested and subjected to point mutagenesis orrecombination before entering the next round of evolution.

We used a chemoenzymatic approach to link substrate A to cells ratherthan a nonspecific chemical conjugation strategy to more reproduciblyarray the substrate on the cell surface and to avoid reagents that mightalter the activity of library members. The B. subtilis Sfpphosphopantetheinyl transferase catalyzes the transfer ofphosphopantetheine from coenzyme A (CoA) onto a specific serine sidechain within an acyl carrier protein or peptide carrier protein. Wechose Sfp to mediate substrate attachment because of its broadsmall-molecule substrate tolerance (3, 21) and its ability toefficiently conjugate phosphopantetheine derivatives to the 12-residueS6 peptide (22) (FIG. 6). We speculated that the small size of the S6peptide would allow it to be well tolerated in the context of the Aga1pmating factor. Functionalized CoA derivatives can be readily prepared byreacting the free thiol of commercially available CoA (3, 21) with acommercially available maleimide-containing bifunctional crosslinker,followed by substrate A bearing a compatible functional group.

To integrate Sfp-catalyzed bioconjugation with yeast display requiredengineering a yeast display vector and yeast strain (FIG. 7). To createa handle for substrate attachment at the cell surface, we fused the S6peptide onto the N-terminus of Aga1p and integrated this construct underthe control of the strong, constitutive GPD promoter in the genome ofSaccharomyces cerevisiae strain BJ5465 (19). We modified the Aga2pexpression construct by inserting the recognition site for tobacco etchvirus (TEV) protease between the hemagglutinin (HA) tag and the codingsequence of the protein of interest. Following incubation of thesubstrate A-conjugated yeast library with substrate B, TEV proteasedigestion removes all library members from the surface, including anyundesired enzymes that bind or react directly with substrate B but donot catalyze A-B bond formation, thus removing a potential source ofundesired background. The HA tag remains on the cell surface and enablesstaining for enzyme display level using an anti-HA antibody. The abilityto efficiently cleave enzymes from the yeast cell surface alsofacilitates enzyme characterization in a cell-free context.

Validation of the Yeast Display System.

Sortase A (srtA) is a sequence-specific transpeptidase found inStaphylococcus aureus and other Gram-positive bacteria. The S. aureusenzyme recognizes an LPXTG (SEQ ID NO: 51) site (X represents any aminoacid), cleaves the scissile amide bond between threonine and glycineusing a nucleophilic cysteine (C184), and resolves the resultingacyl-enzyme intermediate with oligoglycine-linked molecules to generatethe fusion of the LPXT- and oligoglycine-linked peptides or proteins.Sortase A-catalyzed transpeptidation has emerged as a powerful tool forbioconjugation because of the enzyme's high specificity for the LPXTG(SEQ ID NO: 51) motif and its extremely broad substrate toleranceoutside of the recognition elements described above. Because the LPXTG(SEQ ID NO: 51) and oligoglycine motifs can be flanked by virtually anybiomolecule, sortase has been used to label proteins, generate nucleicacid-protein conjugates, and immobilize proteins onto solid supports(23). A significant limitation of srtA is the large quantities of theenzyme or long reaction times that are needed to overcome its poorreaction kinetics (k_(cat)/K_(m LPETG)=200 M⁻¹ s⁻¹; Table 1). Theevolution of a more active S. aureus srtA would therefore significantlyenhance the utility and scope of this bond-forming reaction.

We first examined if yeast-displayed sortases in our system couldcatalyze the reaction between surface-immobilized LPETGG (SEQ ID NO: 33)and exogenous biotinylated triglycine peptide (GGGYK-biotin) (SEQ ID NO:37). To conjugate cells to the LPETGG (SEQ ID NO: 33) substrate, weincubated yeast displaying wild type srtA and the S6 peptide with Sfpand CoA-linked LPETGG (CoA-LPETGG; FIG. 8) (SEQ ID NO: 33). Thesortase-catalyzed reactions were initiated with the addition ofGGGYK-biotin (SEQ ID NO: 37) and 5 mM CaCl₂. After washing, the cellswere stained with streptavidin-PE and an AlexaFluor488-conjugatedanti-HA antibody to analyze the extent of reaction and enzyme displaylevel, respectively, by flow cytometry. When yeast cells displaying wildtype sortase A (WT srtA-yeast) were analyzed, the majority of the cellsexhibited high levels of PE fluorescence, indicating substantialconjugation with GGGYK-biotin (FIG. 2A) (SEQ ID NO: 37). In contrast,wild type srtA-yeast not conjugated to LPETGG (SEQ ID NO: 33), orLPETGG-conjugated (SEQ ID NO: 33) yeast cells displaying the inactiveC184A sortase mutant, exhibited only background levels of PEfluorescence after incubation with GGGYK-biotin (SEQ ID NO: 37),confirming that biotinylation was dependent both on sortase activity andon the presence of both substrates (FIG. 2A).

To verify that enzymes displayed on the yeast cell surface catalyzepseudointramolecular reactions with substrate molecules immobilized onthe same cell, we performed one round of model screening on mixtures ofwild type srtA-yeast and srtA C184A-yeast. Yeast cells were mixed in1:100 and 1:1;000 ratios of wild type: C184A sortases. Each mixture ofcells was coupled with CoA-LPETGG (SEQ ID NO: 33) using Sfp, thenincubated with 50 μM GGGYK-biotin (SEQ ID NO: 37) for 15 min. BecausesrtA binds weakly to GGG (K_(m)=140 μM; Table 1), washing withnonbiotinylated GGG was sufficient to remove any background signal, andTEV digestion was not performed after the reaction. After fluorophorestaining, cells exhibiting both AlexaFluor488 and PE fluorescence wereisolated by FACS (FIG. 2B) and amplified by culturing to saturation. Theplasmid DNA encoding survivors was harvested, and the compositions ofthe recovered genes were analyzed by restriction digestion with HindIIIfollowing PCR amplification. The wild type srtA gene is distinguishablefrom C184A by the presence of an additional HindIII site (FIG. 2C). Inboth model FACS sort experiments, we observed ≧6;000-fold enrichment ofthe wild type gene from both mixtures that were predominantly theinactive C184A mutant (FIG. 2C). Similarly high enrichment factors werealso observed in model sortase screens in which GGG-modified cells werereacted with biotinylated LPETGG (SEQ ID NO: 33) peptide, and in modelbiotin ligase (BirA) screens in which cells displaying a biotinylationsubstrate peptide and wild type BirA were enriched in the presence of alarge excess of cells displaying a less active BirA mutant (FIG. 9).These results show that this system can strongly enrich yeast displayingactive bond-forming enzymes from mixtures containing predominantly yeastdisplaying inactive or less active enzyme variants.

Directed Evolution of Sortase a Enzymes with Improved CatalyticActivity.

Next, we sought to evolve S. aureus srtA for improved activity using theenzyme evolution strategy validated above. We focused on improving thepoor LPXTG (SEQ ID NO: 51) substrate recognition of srtA (K_(m)=7.6 mM;Table 1), which limits the usefulness of sortase-catalyzedbioconjugation by requiring the use of high concentrations of enzyme(>30 μM) or long reaction times to compensate for poor reaction kineticsat the micromolar concentrations of LPXTG (SEQ ID NO: 51) substrate thatare typically used. To direct evolutionary pressure to improve LPXTG(SEQ ID NO: 51) recognition, we formatted the screen such that thetriglycine substrate is immobilized on the cell surface along with theenzyme library, and the biotinylated LPETG (SEQ ID NO: 32) peptide isadded exogenously. This format enables evolutionary pressure forimproved LPETG (SEQ ID NO: 32) recognition to be increased simply bylowering the concentration of LPETG (SEQ ID NO: 32) peptide providedduring the sortase-catalyzed bond-forming reaction.

We randomly mutated the wild type S. aureus srtA gene using PCR withmutagenic dNTP analogs (24) and cloned the resulting genes into themodified yeast display vector using gap repair homologous recombinationto yield a library of 7.8×10⁷ transformants (round 0, R0). Each librarymember contained an average of two nonsilent mutations. The library wassubjected to four rounds of enrichment for sortase activity without anyadditional diversification between rounds. In each round, we subjectedcontrol samples—cells displaying wild type srtA or an improved mutant,or the cells isolated from the previous round—to identical reactionconditions and screening protocols to precisely define FACS gates thatcaptured cells with PE fluorescence corresponding to improved sortaseactivity (FIG. 10). We applied increasing evolutionary pressure forimproved LPETG (SEQ ID NO: 32) recognition by decreasing theconcentration of biotinylated LPETG (SEQ ID NO: 32) substrate 10-foldwith each successive round, starting from 100 μM in the first round andending with 100 nM in the fourth round (FIG. 11). We also increasedevolutionary pressure for overall catalytic activity by accepting asmaller percentage of the most PE-fluorescent cells with each successiveround, ranging from 1.4% in R1 to 0.15% in R4, and by shortening thereaction time in R4 from 60 to 15 min.

To preclude the evolution of specificity for a particularLPETG-containing (SEQ ID NO: 32) sequence, we alternated usingbiotin-LPETGS (SEQ ID NO: 36) (R1 and R3) and biotin-LPETGG (SEQ ID NO:33) (R2 and R4) peptides. After the fourth round of enrichment,surviving genes were subjected to in vitro homologous recombinationusing the NExT procedure (25) and recloned into yeast to yield arecombined and diversified library of 6.9×10⁷ transformants. Theshuffled library (R4Shuf) was subjected to four additional rounds ofsorting (resulting in R5, R6, R7, and R8), with the concentration ofbiotinylated LPETG (SEQ ID NO: 32) peptide dropping from 100 to 10 nM inR8 (FIG. 11).

We developed an assay to rapidly compare the activity of yeast displayedsortase mutants. Yeast cells were incubated with TEV protease to releasethe enzymes from the cell surface into the surrounding supernatant. Thereaction in the supernatant was initiated by the addition of the twopeptide substrates, CoA-LPETGG (SEQ ID NO: 33) and GGGYK-biotin (SEQ IDNO: 37). After 30 min of reaction, Sfp was added to the same reactionmixture to attach the biotinylated adduct and unreacted CoA-LPETGG (SEQID NO: 33) onto the cell surface. We verified that the level ofcell-surface fluorescence after streptavidin-PE staining is a directreflection of the relative amount of biotinylated product in solution(FIG. 12).

We evaluated the mean activity of the yeast pools recovered after eachround of sorting using this assay. Over the course of the selections, weobserved a steady increase in the extent of product formation catalyzedby the recovered sortase mutants. By the last round (R8) the activitysignal was approximately 130-fold greater than that of the initial,unselected library (R0), and approximately 40-fold greater than that ofwild type srtA (FIGS. 3 A and B). These observations suggested that thesystem had evolved sortase variants with substantially improvedactivities.

TABLE 1 Kinetic characterization of mutant sortases k_(cat)/ K_(m)K_(m LPETG), K_(m LPETG), _(GGG-COOH), k_(cat), s⁻¹ mM M⁻¹ s⁻¹ μM WT 1.5± 0.2 7.6 ± 0.5 200 ± 30   140 ± 30 D160N/ 3.7 ± 0.6 1.6 ± 0.4 2,400 ±700  1,200 ± 200 K190E/ K196T (clone 4.2) P94S/ 2.9 ± 0.0 1.1 ± 0.12,600 ± 100  1,700 ± 400 D165A (clone 4.3) P94S/ 4.8 ± 0.8 0.17 ± 0.0328,000 ± 7,000  4,800 ± 700 D160N/ D165A/ K196T P94S/ 4.8 ± 0.6 0.56 ±0.07 8,600 ± 1,500 1,830 ± 330 D160N/ K196T P94S/ 3.8 ± 0.2 0.51 ± 0.387,500 ± 300  1,750 ± 250 D160N/ D165A P94R/ 5.4 ± 0.4 0.23 ± 0.02 23,000± 3,000  2,900 ± 200 D160N/ D165A/ K190E/ K196T P94S 1.6 ± 0.1 2.5 ± 0.6600 ± 200  650 ± 150 D160N 2.3 ± 0.2 3.7 ± 0.5 600 ± 100  330 ± 20 D165A2.4 ± 0.3 3.6 ± 1.0 700 ± 200 1,000 ± 480 K196T 1.2 ± 0.1 3.3 ± 0.8 400± 100  200 ± 70Kinetic parameters k_(cat) and K_(m) were obtained from fitting initialreaction rates at 22.5° C. to the Michaelis-Menten equation. Errorsrepresent the standard deviation of three independent experiments.

Characterization of Evolved Sortase Mutants.

We used the above assay to evaluate the activity of individual clonesfrom R4 and R8 together with wild type srtA and the inactive C184Amutant (FIG. 3B). All tested mutants from R4 exhibited improved activityrelative to wild type, with the two most active mutants, 4.2 and 4.3,showing approximately 20-fold more activity than wild type srtA. Mutantsisolated from R8 exhibited even greater gains in activity, includingfour mutants that were ≧100-fold more active than wild type srtA underthe assay conditions (FIG. 3B).

Sequences of evolved sortase genes revealed the predominance of P94S orP94R, D160N, D165A, and K196T mutations among R8 clones (FIG. 4A andFIG. 13B). Of the 16 unique sequences we isolated from R8, ninecontained all four mutations. Thirteen of the 16 unique sequencescontained at least three of the mutations, and all sequences containedat least two of the four mutations. All of these mutations also appearedin clones isolated from R4, but no clone from R4 contained more than twoof the mutations, suggesting that recombination following R4 enabledcombinations of mutations that persisted in rounds 4-8. Indeed, thehighly enriched tetramutant combination appears to have arisen fromrecombination of two mutations each from clones 4.2 and 4.3, the twomost active mutants isolated from R4. Gene shuffling was therefore animportant component of the evolutionary strategy to generate genesencoding the most active sortases tested.

None of these four mutations have been reported in previous mutationalstudies studying the sortase active site and the molecular basis ofLPETG (SEQ ID NO: 32) substrate recognition (26, 27). To gain insightinto how these mutations improve catalysis, we expressed and purifiedeach sortase single mutant, clones 4.2 and 4.3, and the tetramutant fromEscherichia coli, and we measured the saturation kinetics of wild typesrtA and the mutants using an established HPLC assay (28). The observedkinetic parameters for the wild type enzyme closely match thosepreviously reported (26, 28). Each single mutation in isolationcontributed a small beneficial effect on turnover (k_(cat)) and moresignificant beneficial effects on LPETG (SEQ ID NO: 32) substraterecognition, lowering the K_(m LPETG) up to threefold (Table 1). Theeffects of the mutations in combination were largely additive. Comparedto wild type, 4.2 and 4.3 exhibited a 2.0-2.6-fold improvement ink_(cat) and a 5-7-fold reduction in K_(m LPETG), resulting in anapproximately 15-fold enhancement in catalytic efficiency at using theLPETG (SEQ ID NO: 32) substrate (Table 1). Combining all four mutationsyielded a sortase with a 140-fold improvement in its ability to convertLPETG (SEQ ID NO: 32) (k_(cat)/K_(m LPETG)). This large gain incatalytic efficiency is achieved primarily through 45-fold improvedLPETG (SEQ ID NO: 32) recognition accompanied by a 3-fold gain ink_(cat) (Table 1; FIGS. 14 and 15).

The effects of the individual mutations on LPETG (SEQ ID NO: 32)substrate recognition can be rationalized in light of the reportedsolution structure of wild type S. aureus srtA covalently bound to anLPAT (SEQ ID NO: 39) peptide substrate (29). The mutated residues areall located at the surface of the enzyme, near the LPAT-binding groove(SEQ ID NO: 39) (FIG. 4B). P94 lies at the N terminus of helix 1, andK196 lies at the C terminus of the β7/β8 loop. Both D160 and D165 lie inthe region connecting β6 and β7 that participates in LPETG (SEQ ID NO:32) substrate binding. D165 lies at the N terminus of a 310 helix thatis formed only upon LPAT (SEQ ID NO: 39) binding and makes contacts withthe leucine residue of LPAT (SEQ ID NO: 39). The localization of themutations within loops that line the LPAT (SEQ ID NO: 39) binding groovesuggests that they may be improving binding by altering the conformationof these important loops.

The evolved sortase mutants exhibit decreased GGG substrate binding(Table 1; FIGS. 14 and 15). Compared to wild type, we measured a 30-foldincrease in K_(m GGG) for the sortase A tetramutant. P94S, and D165A hadlarger detrimental effects on K_(m GGG) than D160N and K196T. Theseresults are consistent with mapping of the GGG-binding region proposedby NMR amide backbone chemical shift data. The chemical shifts of thevisible amide hydrogen resonances for residues 92-97 and 165 were amongthe most perturbed upon binding of a Gly3 peptide (29). Because of theabsence of a high-resolution structure of the srtA-Gly3 complex at thistime, it is difficult to rationalize in more detail the basis of alteredK_(m GGG) among evolved mutants.

To recover some of the ability to bind the GGG substrate, we revertedA165 of the tetramutant back to the original aspartic acid residue foundin wild type because our results indicated that the D165A mutation wasmost detrimental for GGG recognition. Compared to the tetramutant, thisP94S/D160N/K196T triple mutant exhibited a 2.6-fold improvement in K_(m)GGG, accompanied by a threefold increase in K_(M LPETG) and no change ink_(cat) (Table 1; FIGS. 14 and 15). We also subjected the R8 yeast poolto one additional round of screening (R9), immobilizing LPETGG (SEQ IDNO: 33) on the cell surface before reaction with 100 nM GGGYK-biotin(SEQ ID NO: 37). The P94S/D160N/K196T reversion mutant was recovered intwo out of the 24 sequenced clones from R9, but a different triplemutant (P94S/D160N/D165A) dominated the R9 population after screening,representing 14/24 sequenced clones (FIG. 13C). Compared to thetetramutant, the K_(m GGG) of this mutant improved by 2.7-fold, whereasthe k_(cat) and

K_(M LPETG) were not altered by more than a factor of 3-fold (Table 1).

We also performed mutagenesis and enrichment to identify additionalmutations that improve GGG recognition in the tetramutant context. Wecombined four R8 clones as templates for additional diversification byPCR, and subjected the resulting yeast library (R8mut) to two rounds ofscreening, immobilizing LPETGG (SEQ ID NO: 33) on the cell surfacebefore reaction with 100-1,000 nM GGGYK-biotin (SEQ ID NO: 37). Aftertwo rounds of enrichment, the K190E mutation originally observed inclone 4.2 was found in 56% of the unique sequenced clones in R10mut, and33% of the clones possessed P94R in place of P94S (FIG. 13D). The otherthree mutations of the tetramutant motif were found intact in 89% of theunique R10mut clones. We constructed the P94R/D160N/D165A/K190E/K196Tpentamutant and assayed its activity. Compared to the tetramutant, theK_(m GGG) of this mutant improved by 1.8-fold, whereas the k_(cat) andK_(M LPETG) were not altered by more than a factor of 1.3-fold. Comparedwith wild type srtA, this pentamutant has a 120-fold higherk_(cat)/K_(M LPETG) and a 20-fold higher K_(m GGG) (Table 1; FIGS. 14and 15). To validate our enzyme kinetics measurements, we followedproduct formation over 1 h and observed turnover numbers of greater than10,000 per hour. The resulting data (FIG. 16) yielded k_(cat) andK_(M LPETG) values that closely agree with our kinetics measurements(Table 1). These results indicate that relatives of the evolvedtetramutant can exhibit partially restored GGG binding and thereforeprovide alternative enzymes for applications in which the GGG-linkedsubstrate is available only in limited quantities.

Cell-Surface Labeling with an Evolved Sortase.

The improved activities of the evolved sortases may enhance theirutility in bioconjugation applications such as the site-specificlabeling of LPETG-tagged (SEQ ID NO: 21) proteins expressed on thesurface of living cells. In these applications, the effectiveconcentration of the LPETG (SEQ ID NO: 21) peptide is typically limitedto micromolar or lower levels by endogenous expression levels, andtherefore the high K_(M LPETG) of wild type srtA (K_(M LPETG)=7.6 mM;Table 1) necessitates the use of a large excess of coupling partner andenzyme to drive the reaction to a reasonable yield. Because it istypically straightforward to synthesize milligram quantities of shortoligoglycine-linked probes using solid-phase peptide chemistry, wehypothesized that the much higher k_(cat)/K_(M LPETG) of the evolvedsortases might enable them to mediate cell-surfacing reactions thatwould be inefficient using the wild type enzyme.

We expressed human CD 154 tagged with the LPETG (SEQ ID NO: 32) sequenceat its C terminus on the surface of HeLa cells and compared the labelingof the live cells with GGGYK-biotin (SEQ ID NO: 37) using wild type srtAand the evolved P94S/D160N/K196T mutant. After staining with astreptavidin-AlexaFluor594 conjugate, flow cytometry analysis revealedthat the evolved sortase yielded ≧30-fold higher median fluorescencethan the wild type enzyme (FIG. 5A). Although we used conditions similarto those used to label HEK293 cells using wild type srtA forfluorescence microscopy (4), over four independent replicates, the wildtype enzyme did not result in fluorescence more than 2.8-fold higherthan the background fluorescence of cells incubated in the absence ofenzyme (FIG. 5A). Consistent with the flow cytometry data, live-cellfluorescence microscopy confirmed very weak labeling by wild type srtAand much more efficient labeling by the evolved sortase mutant (FIG.5B). Cells expressing CD154 without the LPETG (SEQ ID NO: 32) tag werenot labeled to a significant extent by the evolved sortase, indicatingthat the site-specificity of the enzyme has not been significantlycompromised. Under the conditions tested, the evolved sortase triple,tetra-, and pentamutants all exhibit comparable and efficientcell-surface labeling, despite their differences in K_(m GGG) (FIG. 17).Collectively, our results suggest that the sortase variants evolvedusing the enzyme evolution system developed in this work aresubstantially more effective than the wild type enzyme at labelingLPETG-tagged (SEQ ID NO: 32) proteins on the surface of live mammaliancells.

Directed Evolution of Sortase a Enzymes with Altered SubstrateSpecificity.

In an effort to develop S. Aureus Sortase A derivatives of alteredsubstrate specificity, we took advantage of the single turnover natureof the selection described herein. By co-incubating yeast displaying aSortase A variant with both biotinylated LPESG (SEQ ID NO: 38) and alarge excess of unbiotinylated LPETG (SEQ ID NO: 32), we successfullyenriched for enzymes with reduced affinity for LPETG (SEQ ID NO: 32),and increased affinity for LPESG (SEQ ID NO: 38). Beginning from a naïvelibrary of >10⁷ variants of the pentamutant identified previously, weperformed serial selections for the ability to react with LPESG (SEQ IDNO: 38) in the presence of competitive LPETG (SEQ ID NO: 32) (FIGS.19-22). After four rounds of FACS sorting, we sequenced the remainingclones, observing complete convergence to a single variant, identifiedas Sortase 4S.4 and consisting of the 15 mutations P86L, P94R, N98S,A104T, A118T, F122S, D124G, N127S, K134R, D160N, D165A K173E, K177E,K190E, and K196T relative to wild type S. Aureus Sortase A. Furtherselections attempted to revert as many of these mutations as possible,leading ultimately to the optimized variant 4S.12.2, consisting ofmutations P94R, N98S, A104T, A118T, F122S, D124G, K134R, D160N, D165A,K173E, K177E, K190E, and K196T. On characterization, both variantsshowed completely abrogated activity against LPETG (SEQ ID NO: 32), andactivity against LPESG (SEQ ID NO: 38) comparable to the starting typepentamutant.

Discussion

Yeast display, Sfp-catalyzed bioconjugation, and cell sorting wereintegrated into a general directed evolution strategy for enzymes thatcatalyze bond-forming reactions. The system was validated through modelselections enriching for S. aureus sortase A-catalyzed transpeptidationactivity, attaining enrichment factors greater than 6,000 after a singleround of sorting. The system was applied to evolve sortase A forimproved catalytic activity. After eight rounds of sorting with oneintermediate gene shuffling step, sortase A variants were isolated thatcontained four mutations that together resulted in a 140-fold increasein LPETG-coupling (SEQ ID NO: 32) activity compared with the wild typeenzyme. An evolved sortase enabled much more efficient labeling ofLPETG-tagged (SEQ ID NO: 32) human CD154 expressed on the surface ofHeLa cells compared with wild type sortase.

The kinetic properties of the mutant sortases accurately reflected thescreening strategy. The 50-fold decrease in K_(M LPETG) of thetetramutant compared to wild type is consistent with lowering theconcentration of free biotinylated LPETG (SEQ ID NO: 32) peptide duringthe reaction in successive rounds. Meanwhile, this screening formatensured that a high effective molarity of GGG was presented to eachenzyme candidate over eight rounds of enrichment, which was estimated tobe approximately 950 μM (FIG. 18). GGG recognition among evolvedsortases drifted during evolution. The threefold increase in k_(cat) ofthe tetramutant compared to that of the wild type enzyme may haveresulted from screening pressures arising from shortening the reactiontime in later rounds. Larger increases in k_(cat) may require modifiedselection or screening strategies that explicitly couple survival withmultiple turnover kinetics, perhaps by integrating our system with invitro compartmentalization.

Despite the widespread use of yeast display in the evolution of bindinginteractions (18), sortase A is only the third enzyme to be evolvedusing yeast display, in addition to horseradish peroxidase (30, 31) andan esterase catalytic antibody (32). The results described hereinhighlight the advantageous features of yeast display for enzymeevolution, including quality control mechanisms within the secretorypathway that ensure display of properly folded proteins andcompatibility with FACS (18). As an alternative to yeast or other cellsas the vehicle for display, M13 phage simultaneously displaying an Sfppeptide substrate and an enzyme library may also be used (33). As themethodology disclosed herein does not rely on any particular screenableor selectable property of the substrates or product, it is in principlecompatible with any bond-forming enzyme that can be expressed in a cell,e.g., in yeast, including glycosylated proteins that are likelyincompatible with phage and mRNA display, provided that linkage of thesubstrates to CoA and to the affinity handle is possible and toleratedby the enzyme or its evolved variants. In cases in which the enzymeaccepts only one of these modifications, product-specific antibodies inprinciple could be used to detect bond formation. Furthermore,integrating the yeast display system provided herein with the multicolorcapabilities of FACS enables the evolution of enzyme substratespecificity.

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All publications, patents and sequence database entries mentionedherein, including those items listed in the Summary, Brief Descriptionof the Drawings, Detailed Description, and Examples sections, are herebyincorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of conflict, the present application,including any definitions herein, will control.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the claims or from relevant portions of the description isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

What is claimed is:
 1. A sortase comprising an amino acid sequence thatis at least 90% identical to the amino acid sequence of Staphylococcusaureus (S. aureus) Sortase A as provided as SEQ ID NO: 1 or to aminoacid residues 1-150 of SEQ ID NO: 21, wherein the amino acid sequence ofthe sortase comprises two or more mutations selected from the groupconsisting of P94S, P94R, E106G, F122Y, K154R, D160N, D165A, G174S,K190E, and K196T.
 2. The sortase of claim 1, wherein the sortasecomprises an amino acid sequence that is at least 95% identical to SEQID NO: 1 or to amino acid residues 1-150 of SEQ ID NO:
 21. 3. Thesortase of claim 1, wherein the amino acid sequence of the sortasecomprises at least three mutations selected from the group consisting ofP94S, P94R, E106G, F122Y, K154R, D160N, D165A, G174S, K190E, and K196T.4. The sortase of claim 1, wherein the sortase comprises a P94S or P94Rmutation, a D160N mutation, a D165A mutation, a K190E mutation, and aK196T mutation.
 5. The sortase of claim 1, wherein the sortase comprisesa P94S or P94R mutation, a D160N mutation, and a K196T mutation.
 6. Thesortase of claim 1, wherein the sortase comprises a P94S or P94Rmutation, a D160N mutation, and a D165A mutation.
 7. The sortase ofclaim 1, wherein the sortase comprises a P94S or P94R mutation, a D160Nmutation, a D165A mutation, and a K196T mutation.
 8. The sortase ofclaim 1, wherein the sortase exhibits a k_(cat) that is at least1.5-fold greater than the k_(cat) of the Sortase A having the amino acidsequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ IDNO:
 21. 9. The sortase of claim 1, wherein the sortase exhibits a K_(M)for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32)that is at least 2-fold-less than the K_(M) of the Sortase A having theamino acid sequence provided as SEQ ID NO: 1 or amino acid residues1-150 of SEQ ID NO:
 21. 10. The sortase of claim 1, wherein the sortaseexhibits a K_(M) for a substrate comprising the amino acid sequence GGGthat is not more than 2-fold greater than the K_(M) of the Sortase Ahaving the amino acid sequence provided as SEQ ID NO: 1 or amino acidresidues 1-150 of SEQ ID NO:
 21. 11. The sortase of claim 1, wherein thesortase exhibits a ratio of K_(cat)/K_(M) for a substrate comprising theamino acid sequence LPETG (SEQ ID NO: 32) that is least 2-fold greaterthan the K_(cat)/K_(M) ratio of the Sortase A having the amino acidsequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ IDNO:
 21. 12. A sortase catalyzing transpeptidation of substrates otherthan LPETG (SEQ ID NO: 32), the sortase comprising an amino acidsequence that is at least 90% identical to the amino acid sequence of S.aureus Sortase A as provided as SEQ ID NO: 1 or to amino acid residues1-150 of SEQ ID NO: 21, wherein the amino acid sequence comprises two ormore mutations selected from the group consisting of P86L, P94R, N98S,A104T, A118T, F122S, D124G, N127S, K134R, D160N, D165A K173E, K177E,K190E, and K196T.
 13. The sortase of claim 12, wherein the substratecomprises the amino acid sequence LPXS, LAXT, MPXT, LAXS, NPXT, NAXT,NAXS, LPXP, or LPXTA (SEQ ID NO: 40), wherein each occurrence of Xrepresents independently any amino acid residue.
 14. The sortase ofclaim 12, wherein the substrate comprises the amino acid sequence LPESG(SEQ ID NO: 38).
 15. A method for transpeptidation, the methodcomprising contacting the sortase of claim 1 with a substrate comprisingan LPETG (SEQ ID NO: 32) sequence and with a substrate comprising a GGGsequence under conditions suitable for sortase-mediatedtranspeptidation.
 16. The method of claim 15, wherein the LPETG (SEQ IDNO: 32) substrate and/or the GGG substrate are on the surface of a cell.17. The method of claim 16, wherein the cell expresses a surface markerprotein that is C-terminally fused to an LPETG (SEQ ID NO: 32) sequence.18. The method of claim 16, wherein the cell expresses a surface markerprotein that is N-terminally fused to a GGG sequence.
 19. The method ofclaim 15, wherein the LPETG (SEQ ID NO: 32) substrate and/or the GGGsubstrate are polypeptides or proteins, and wherein the method resultsin the generation of a protein fusion.
 20. The method of claim 15,wherein the LPETG (SEQ ID NO: 32) substrate or the GGG substratecomprises a non-protein structure.
 21. The sortase of claim 1, whereinthe sortase comprises an amino acid sequence that is at least 98%identical to SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO:21.
 22. The sortase of claim 1, wherein the sortase comprises an aminoacid sequence that is at least 99% identical to SEQ ID NO: 1 or to aminoacid residues 1-150 of SEQ ID NO:
 21. 23. The sortase of claim 1,wherein the amino acid sequence of the sortase comprises at least fourmutations selected from the group consisting of P94S, P94R, E106G,F122Y, F154R, D160N, D165A, G174S, K190E, and K196T.
 24. The sortase ofclaim 1, wherein the sortase exhibits a K_(M) for a substrate comprisingthe amino acid sequence LPETG (SEQ ID NO: 32) that is at least 5-foldless than the K_(M) of the Sortase A having the amino acid sequenceprovided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.25. The sortase of claim 1, wherein the sortase exhibits a K_(M) for asubstrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) thatis at least 10-fold less than the K_(M) of the Sortase A having theamino acid sequence provided as SEQ ID NO: 1 or of the Sortase A havingthe amino acid sequence provided by amino acid residues 1-150 of SEQ IDNO:
 21. 26. The sortase of claim 1, wherein the sortase exhibits a K_(M)for a substrate comprising the amino acid sequence GGG that is not morethan 5-fold greater than the K_(M) of the Sortase A having the aminoacid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 ofSEQ ID NO:
 21. 27. The sortase of claim 1, wherein the sortase exhibitsa K_(M) for a substrate comprising the amino acid sequence GGG that isnot more than 10-fold greater than the K_(M) of the Sortase A having theamino acid sequence provided as SEQ ID NO: 1 or amino acid residues1-150 of SEQ ID NO:
 21. 28. The sortase of claim 1, wherein the sortaseexhibits a ratio of K_(cat)/K_(M) for a substrate comprising the aminoacid sequence LPETG (SEQ ID NO: 32) that is least 5-fold greater thanthe K_(cat)/K_(M) ratio of the Sortase A having the amino acid sequenceprovided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.29. The sortase of claim 1, wherein the sortase exhibits a ratio ofK_(cat)/K_(M) for a substrate comprising the amino acid sequence LPETG(SEQ ID NO: 32) that is least 10-fold greater than the K_(cat)/K_(M)ratio of the Sortase A having the amino acid sequence provided as SEQ IDNO: 1 or amino acid residues 1-150 of SEQ ID NO:
 21. 30. The sortase ofclaim 1, wherein the sortase exhibits a ratio of K_(cat)/K_(m) for asubstrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) thatis least 50-fold greater than the K_(cat)/K_(M) ratio of the Sortase Ahaving the amino acid sequence provided as SEQ ID NO: 1 or amino acidresidues 1-150 of SEQ ID NO:
 21. 31. The sortase of claim 1, wherein thesortase has an amino acid sequence provided by a sequence selected fromthe group consisting of SEQ ID NOs: 17, 18, 19, 20, 26, 27, 28, 29, 30,and
 31. 32. The method of claim 20, wherein the LPETG substrate (SEQ IDNO: 32) or the GGG substrate comprises a detectable label, a smallmolecule, a nucleic acid, or a polysaccharide.